Method for the in vivo synthesis of 4-hydroxymethylfurfural and derivatives thereof

ABSTRACT

The present disclosure provides recombinant microorganisms and methods for the production of 4-HMF, 2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid, 2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and/or 2,4-FDCA from a carbon source. The method provides for engineered microorganisms that express endogenous and/or exogenous nucleic acid molecules that catalyze the conversion of a carbon source into 4-HMF, 2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid, 2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and/or 2,4-FDCA. The disclosure further provides methods of producing polymers derived from 4-HMF, 2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid, 2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and/or 2,4-FDCA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/806,728, filed Mar. 2, 2020, entitled “METHOD FOR THE IN VIVOSYNTHESIS OF 4-HYDROXYMETHYLFURFURAL AND DERIVATIVES THEREOF”, whichclaims priority to U.S. Provisional Application No. 62/812,904 filedMar. 1, 2019, entitled “METHOD FOR THE IN VIVO SYNTHESIS OF4-HYDROXYMETHYLFURFURAL AND DERIVATIVES THEREOF”, the entire disclosuresof which are incorporated by reference herein.

FIELD OF THE INVENTION

This application relates to recombinant microorganisms for thebiosynthesis of one or more of 4-HMF, 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and 2,4-FDCAand methods of producing the recombinant microorganisms. The applicationalso relates to methods of producing one or more of 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and 2,4-FDCAwith enzymatic catalysts in the absence of microorganisms orsubstantially free of microorganisms. The application further relates tomethods of producing a polymer and a plasticizer agent from one or moreof 2,4-furandimethanol, furan-2,4-dicarbaldehyde,4-(hydroxymethyl)furoic acid, 2-formylfuran-4-carboxylate,4-formylfuran-2-carboxylate, and 2,4-FDCA. The application furtherrelates to compositions comprising one or more of these compounds and/orthe recombinant microorganisms.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 127125-5014-US-01_Sequence_Listing_ST25.txt. Thetext file is about 241 KB, was created on Jan. 29, 2021, and is beingsubmitted electronically via EFS-Web.

BACKGROUND

2,5-Furandicarboxylic acid (2,5-FDCA) has gained much attention due toits potential of substituting terephthalic acid in the synthesis ofpolyesters, specially polyethylene terephthalate (PET) (Sousa, AndreiaF., et al. “Biobased polyesters and other polymers from 2,5-furandicarboxylic acid: a tribute to furan excellency.” Polymerchemistry 6.33 (2015): 5961-5983). Substituting terephthalic acid to itsfuran analogue 2,5-FDCA in PET can lead to 2,5-furandicarboxylate(2,5-PEF) and this polymer has several advantages when compared to PET.In one aspect, 2,5-PEF has better thermal, barrier and mechanicalproperties when compared to its counterpart (PEP Report 294).Furthermore, as it is known that ethylene glycol could be produced fromrenewable resources, then 2,5-PEF could be 100% renewable as opposed tothe semi renewable PET.

Despite all the aforementioned advantages of 2,5-FDCA in comparison toterephthalic acid, 2,5-FDCA production cost is still a currentlimitation in expanding the monomer usage. Existing technologies are notcost-competitive when compared to terephthalic acid. One of the possiblereasons for this is related to the several sequential industrial stepsrequired. One issue that could help reduce 2,5-FDCA production costs isfinding a direct fermentation route from sugar to the desired molecule,but such a route has never been reported.

The present disclosure a direct fermentation pathway for 2,4-FDCA, anisomer of 2,5-FDCA. To our knowledge, besides the present disclosure,there is no described direct fermentation routes for any of FDCAisomers.

Significantly, the disclosed 2,4-FDCA molecule possesses uniqueproperties compared to the well-studied 2,5-FDCA. Catalyticallypolymerizing 2,4-FDCA with a diol yields a polymer composed of 2,4-FDCAwith valuable properties. In one study, Thiyagarajan and collaborators(2014) compare polyesters made of 2,4-FDCA, 3,4-FDCA, 2,5-FDCA andterephthalic acid and concluded that 2,4-FDCA and 3,4-FDCA polyesterscan be made in sufficient molecular weights by industrially applicablemethods (Thiyagarajan, Shanmugam, et al. “Biobased furandicarboxylicacids (FDCAs): effects of isomeric substitution on polyester synthesisand properties.” Green Chemistry 16.4 (2014): 1957-1966). In anotherstudy, Thiyagarajan and colleagues concluded that structural analysis of2,4-FDCA and 2,5-FDCA reveal that 2,4-FDCA possesses more linearcharacteristics resembling terephthalic acid than does 2,5-FDCA. Thesefeatures make 2,4-FDCA an interesting monomer for synthetic polyesters(Thiyagarajan et al. “Concurrent formation of furan-2,5- andfuran-2,4-dicarboxylic acid: unexpected aspects of the Henkel reaction”RSC Advances 3 (2013): 15678-15686). Further, these materials haveproperties unlike 2,5-FDCA polyesters (Bourdet et al. “MolecularMobility in Amorphous Biobased Poly (ethylene 2, 5-furandicarboxylate)and Poly (ethylene 2, 4-furandicarboxylate).” Macromolecules 51.5(2018): 1937-1945).

In certain cases, 2,4-FDCA polymers have been reported to have superiorproperties to those possessed by 2,5-FDCA polymers. Cui andcollaborators (2016) report that the bond-angle between the doublecarboxyl groups linking with the central ring is a key factor thatinfluences the stability of nematic liquid crystal molecules such asthose utilized in LCD TVs, notebook computers, and other displayelements (Cui, Min-Shu, et al. “Production of 4-hydroxymethylfurfuralfrom derivatives of biomass-derived glycerol for chemicals andpolymers.” ACS Sustainable Chemistry & Engineering 4.3 (2016):1707-1714). The first discovered liquid crystal, terephthalic aciddiester molecules has a bond-angle between two carboxyl groups of 180°.In comparison, 2,5-furan dicarboxylic acid has a bond-angle between twocarboxyl groups of 137°. Significantly, 2,4-furan dicarboxylic acid hasa bond-angle between two carboxyl groups of 160° making it more suitablefor synthesis of nematic liquid crystal molecules.

Despite these potential applications of 2,4-FDCA polymers, theproduction cost of 2,4-FDCA is also a current bottleneck in expandingthis monomer applications (Cui M S, et al. (2016) Production of4-Hydroxymethylfurfural from Derivatives of Biomass-Derived Glycerol forChemicals and Polymers. ACS Sustainable Chem. Eng. 4(3):1707-1714 andWO2011003300A1). Previous synthesis of 2,4-substituted furans, including2,4-FDCA, required multiple synthetic steps and therefore2,4-FDCA-derived polymers are cost-prohibitive by currently availablemethodologies and industrial techniques.

The present disclosure provides, for the first time, a directfermentation route to 2,4-FDCA in a recombinant microorganism. The noveldirect fermentation of 2,4-FDCA from a glyceraldehyde-3-phosphate (G3P)from a carbon feedstock such as glucose, xylose, glycerol, or from anyCO2 derived/capture technology will enable the production of novelpolymers and materials with commercial applicability on an industrialscale. The present disclosure further provides, for the first time,direct fermentation routes for the production of one or more of 4-HMF,2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoicacid, 2-formylfuran-4-carboxylate, and 4-formylfuran-2-carboxylate in arecombinant microorganism. The present disclosure also demonstrate, forthe first time, that endogenous phosphatases from yeast and E. coli areable to dephosphorylate (5-formylfuran-3-yl)methyl phosphate to 4-HMFand that enzymes (oxidases, dehydrogenase and/or peroxigenase) arecapable to oxidize the 4-HMF to 2,4 FDCA (directly or through theproduction of intermediates). While some of the enzymes candidates heredeployed have been characterized as having activity on a 5-HMF isomersubstrate, and intermediates, their activity against 4-HMF (and itsintermediates) has nto been characterized. These novel directfermentation routes will enable the production of 2,4-substituted furanswith commercial applicability. See Deng et al. (2013. Linked Strategyfor the Production of Fuels via Formose Reaction. Scientific Reports,3:1244) for exemplary applications of 4-HMF as a precursor to biofuels.See Zeng et al. (2013. Bio-based Furan Polymers with Self-HealingAbility. Macromolecules, 46.5:1794-1802) for exemplary applications of2,4-furandimethanol in polymers with advanced properties.

SUMMARY OF THE DISCLOSURE

In certain cases, 2,4-FDCA polymers have been reported to have superiorproperties to those possessed by 2,5-FDCA polymers. Cui andcollaborators (2016) report that the bond-angle between the doublecarboxyl groups linking with the central ring is a key factor thatinfluences the stability of nematic liquid crystal molecules such asthose utilized in LCD TVs, notebook computers, and other displayelements (Cui, Min-Shu, et al. “Production of 4-hydroxymethylfurfuralfrom derivatives of biomass-derived glycerol for chemicals andpolymers.” ACS Sustainable Chemistry & Engineering 4.3 (2016):1707-1714). The first discovered liquid crystal, terephthalic aciddiester molecules has a bond-angle between two carboxyl groups of 180°.In comparison, 2,5-furan dicarboxylic acid has a bond-angle between twocarboxyl groups of 137°. Significantly, 2,4-furan dicarboxylic acid hasa bond-angle between two carboxyl groups of 160° making it more suitablefor synthesis of nematic liquid crystal molecules.

The disclosure provides a method of producing 2,4-furandicarboxylic acid(2,4-FDCA) by enzymatically converting glyceraldehyde 3-phosphate (G3P)to 2,4-furandicarboxylic acid (2,4-FDCA), the method comprising: (a)providing G3P in the presence of a methyl phosphate synthase thatcatalyzes the conversion of G3P to (5-formylfuran-3-yl)methyl phosphate;(b) providing the (5-formylfuran-3-yl)methyl phosphate from (a) aphosphatase that catalyzes the conversion of the(5-formylfuran-3-yl)methyl phosphate to 4-hydroxymethylfurfural (4-HMF);(c) providing the 4-HMF from (b) to a dehydrogenase and/or an oxidasethat catalyzes independently or in synergy the oxidation of 4-HMF from(b) to 2,4 FDCA, directly or through the production of intermediatesfuran-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,4-formylfuran-2-carboxylate, 4-formylfuran-2-carboxylate,2-formylfuran-4-carboxylate.

In some embodiments, the 2,4-FDCA is produced fromfuran-2,4-dicarbaldehyde, and/or-(hydroxymethyl)furoic acidintermediates, wherein: (a) a dehydrogenase, an oxidase, or aperoxigenase catalyzes the conversion of the 4-HMF tofuran-2,4-dicarbaldehyde, and/or 4-(hydroxymethyl)furoic acid; and/or(b) a dehydrogenase, an oxidase, or a peroxigenase catalyzes theconversion of the furan-2,4-dicarbaldehyde from (a) to4-formylfuran-2-carboxylate; and/or (c) a dehydrogenase, an oxidase, ora peroxigenase catalyzes the conversion of the 4-(hydroxymethyl)furoicacid from (a) to 4-formylfuran-2-carboxylate; and/or (d) adehydrogenase, an oxidase, or a peroxigenase catalyzes the conversion ofthe furan-2,4-dicarbaldehyde from (a) to 2-formylfuran-4-carboxylate;and or (e) a dehydrogenase, an oxidase, or a peroxigenase catalyzes theconversion of the the 4-formylfuran-2-carboxylate from (b) and/or (c) orthe 2-formylfuran-4-carboxylate from (d) to 2,4-FDCA.

In some embodiments, the methyl phosphate synthase from (a) isclassified as EC number 4.2.3.153. In some embodiments, the methylphosphate synthase is (5-formylfuran-3-yl)methyl phosphate synthase. Insome embodiments, the (5-formylfuran-3-yl)methyl phosphate synthase isselected from MfnB1, MfnB7, and MfnB14.

In some embodiments, the (5-formylfuran-3-yl)methyl phosphate synthasecomprises an amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO: 7,or SEQ ID NO: 14. In some embodiments, the phosphatase from (b) isclassified as EC number 3.1.3. In some embodiments, the phosphatase isclassified as a haloacid dehalogenase. In some embodiments, thephosphatase is endogenous to the host.

In some embodiments, the phosphatase enzyme endogenous to the host isoverexpressed. In some embodiments, wherein the phosphatase is a 4-HMFphosphatase.

In some embodiments, the 4-HMF phosphatase is derived from Streptomycescoelicolor, Saccharomyces cerevisiae, or Escherichia coli.

In some embodiments, the 4-HMF phosphatase is encoded by an amino acidsequence comprising SEQ ID NO: 28, any one of SEQ ID NOs 40-52, or anyone of SEQ ID NOs 53-68.

In some embodiments, the dehydrogenase from (c) is classified as ECnumber 1.1.1. or EC number 1.2.1. In some embodiments, the dehydrogenaseis an alcohol dehydrogenase or an aldehyde dehydrogenase. In someembodiments, the oxidase from (c) is classified as EC number 1.1.3. Insome embodiments, the oxidase is 5-hydroxymethylfurfural oxidase. Insome embodiments, the dehydrogenase is classified as EC number 1.2.1. orEC number 1.1.1. In some embodiments, the dehydrogenase is an aldehydedehydrogenase or and alcohol dehydrogenase.

In some embodiments, the oxidase is classified as EC number 1.1.3. Insome embodiments, the oxidase is 5-hydroxymethylfurfural oxidase. Insome embodiments, the oxidase is a 4-HMF oxidase. In some embodiments,the 4-HMF oxidase is selected from HmfH6 and HmfH7. In some embodiments,the 4-HMF oxidase comprises an amino acid sequence comprising SEQ ID NO:85 or SEQ ID NO: 86.

In some embodiments, the dehydrogenase is classified as EC number 1.2.1.In some embodiments, the dehydrogenase is an aldehyde dehydrogenase.

The disclosure provides a recombinant microorganism capable of producing2,4-furandicarboxylic acid (2,4-FDCA) from a feedstock comprising acarbon source, wherein the recombinant microorganism expresses thefollowing: (a) endogenous and/or exogenous nucleic acid moleculescapable of converting a carbon source to glyceraldehyde 3-phosphate(G3P); (b) at least one endogenous or exogenous nucleic acid moleculeencoding a (5-formylfuran-3-yl)methyl phosphate synthase that catalyzesthe conversion of G3P from (a) to (5-formylfuran-3-yl)methyl phosphate;(c) at least one endogenous or exogenous nucleic acid molecule encodinga phosphatase that catalyzes the conversion of(5-formylfuran-3-yl)methyl phosphate from (b) to 4-hydroxymethylfurfural(4-HMF); (d) at least one endogenous or exogenous nucleic acid moleculeencoding a peroxigenase, dehydrogenase, or a oxidase that catalyzesindependently or in synergy the oxidation of 4-HMF from (c) to 2,4 FDCA,directly or through the production of intermediatesfuran-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,4-formylfuran-2-carboxylate, 4-formylfuran-2-carboxylate,2-formylfuran-4-carboxylate.

In some embodiments, the carbon source comprises a hexose, a pentose,glycerol, CO2, sucroses and/or combinations thereof. In someembodiments, the methyl phosphate synthase from (a) is classified as ECnumber 4.2.3.153. In some embodiments, wherein the synthase is(5-formylfuran-3-yl)methyl phosphate synthase.

In some embodiments, the phosphatase from (c) is classified as EC number3.1.3. In some embodiments, the phosphatase is classified as haloaciddehalogenase. In some embodiments, the phosphatase is endogenous to thehost. In some embodiments, phosphatase enzyme endogenous to the host isoverexpressed.

In some embodiments, the oxidase from (d) is classified as EC number1.1.3. In some embodiments, the oxidase from (d) is a5-hydroxymethylfurfural oxidase.

In some embodiments, the dehydrogenase from (d) is classified as ECnumber 1.1.1. or EC number 1.2.1. In some embodiments, the dehydrogenaseis an alcohol dehydrogenase or an aldehyde dehydrogenase.

In some embodiments, the 2,4-FDCA is produced fromfuran-2,4-dicarbaldehyde, and/or -(hydroxymethyl)furoic acidintermediates, wherein: (a) a dehydrogenase, an oxidase, or aperoxigenase catalyzes the conversion of the 4-HMF from (c) tofuran-2,4-dicarbaldehyde, and/or 4-(hydroxymethyl)furoic acid; and/or(b) a dehydrogenase, an oxidase, or a peroxigenase catalyzes theconversion of the furan-2,4-dicarbaldehyde from (a) to4-formylfuran-2-carboxylate; and/or (c) a dehydrogenase, an oxidase, ora peroxigenase catalyzes the conversion of the 4-(hydroxymethyl)furoicacid from (b) to 4-formylfuran-2-carboxylate; and/or (d) adehydrogenase, an oxidase, or a peroxigenase catalyzes the conversion ofthe furan-2,4-dicarbaldehyde from (c) to 2-formylfuran-4-carboxylate;and/or (e) a dehydrogenase, an oxidase, or a peroxigenase catalyzes theconversion of the 4-formylfuran-2-carboxylate from (b) and/or (c) or the2-formylfuran-4-carboxylate from (d) to 2,4-FDCA.

In some embodiments, the dehydrogenase from (a), (b), (c), (d) and/or(e) is classified as EC number 1.2.1. or EC number 1.1.1 In someembodiments, the dehydrogenase is an aldehyde dehydrogenase or analcohol dehydrogenase. In some embodiments, the oxidase from (a), (b),(c), (d) and/or (e) is classified as EC number 1.1.3. In someembodiments, the oxidase is 5-(hydroxymethyl)furfural oxidase.

In some embodiments, the one or more recombinant microorganisms arederived from a parental microorganism selected from the group consistingof Clostridium sp., Clostridium ljungdahlii, Clostridiumautoethanogenum, Clostridium ragsdalei, Eubacterium limosum,Butyribacterium methylotrophicum, Moorella thermoacetica,Corynebacterium glutamicum, Clostridium aceticum, Acetobacterium woodii,Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans,Clostridium formicoaceticum, Clostridium scatologenes, Moorellathermoautotrophica, Acetonema longum, Blautia producta, Clostridiumglycolicum, Clostridium magnum, Candida krusei, Clostridium mayombei,Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridiumbeijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusaovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum,Issatchenkia orientalis, Sporomusa termitida, Moorella glycerini,Eubacterium aggregans, Treponema azotonutricium, Pichia kudriavzevii,Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillussp, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis,and Terrisporobacter glycolicus.

In some embodiments, the one or more recombinant microorganisms arederived from a parental microorganism selected from the group consistingof Clostridium sp., Clostridium ljungdahlii, Clostridiumautoethanogenum, Clostridium ragsdalei, Eubacterium limosum,Butyribacterium methylotrophicum, Moorella thermoacetica,Corynebacterium glutamicum, Clostridium aceticum, Acetobacterium woodii,Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans,Clostridium formicoaceticum, Clostridium scatologenes, Moorellathermoautotrophica, Acetonema longum, Blautia producta, Clostridiumglycolicum, Clostridium magnum, Candida krusei, Clostridium mayombei,Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridiumbeijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusaovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum,Issatchenkia orientalis, Sporomusa termitida, Moorella glycerini,Eubacterium aggregans, Treponema azotonutricium, Pichia kudriavzevii,Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillussp, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis,and Terrisporobacter glycolicus.

The disclosure provides a method of producing 2,4-FDCA using arecombinant microorganism of claim 27, the method comprising cultivatingthe recombinant microorganism in a culture medium containing a feedstockproviding a carbon source until the 2,4-FDCA is produced.

The disclosure provides a method of producing a recombinantmicroorganism capable of producing 2,4-FDCA from a feedstock comprisinga carbon source, the method comprising introducing into and/oroverexpressing in the recombinant microorganism the following: (a)endogenous and/or exogenous nucleic acid molecules capable of convertingglycerol or a monosaccharide to glyceraldehyde 3-phosphate (G3P); (b) atleast one endogenous or exogenous nucleic acid molecule encoding a(5-formylfuran-3-yl)methyl phosphate synthase that catalyzes theconversion of G3P from (a) to (5-formylfuran-3-yl)methyl phosphate; (c)at least one endogenous or exogenous nucleic acid molecule encoding aphosphatase that catalyzes the conversion of (5-formylfuran-3-yl)methylphosphate from (b) to 4-hydroxymethylfurfural (4-HMF); (d) at least oneendogenous or exogenous nucleic acid molecule encoding a peroxigenase,dehydrogenase, or a oxidase that catalyzes independently or in synergythe oxidation of 4-HMF from (c) to 2,4 FDCA, directly or through theproduction of intermediates furan-2,4-dicarbaldehyde,4-(hydroxymethyl)furoic acid, 4-formylfuran-2-carboxylate,4-formylfuran-2-carboxylate, 2-formylfuran-4-carboxylate.

In some embodiments, the carbon source comprises a hexose, a pentose,glycerol, CO2, sucroses and/or combinations thereof. In someembodiments, the methyl phosphate synthase from (a) is classified as ECnumber 4.2.3.153. In some embodiments, the synthase is(5-formylfuran-3-yl)methyl phosphate synthase. In some embodiments, thephosphatase from (c) is classified as EC number 3.1.3. In someembodiments, the phosphatase is classified as haloacid dehalogenase. Insome embodiments, the phosphatase is endogenous to the host. In someembodiments, the phosphatase enzyme endogenous to the host isoverexpressed.

In some embodiments, the dehydrogenase from (d) is classified as ECnumber 1.1.1. or EC number 1.2.1. In some embodiments, the dehydrogenaseis an alcohol dehydrogenase or an aldehyde dehydrogenase. In someembodiments, the oxidase from (d) is classified as EC number 1.1.3. Insome embodiments, the oxidase is (5-(hydroxymethyl)furfural oxidase.

In some embodiments, the 2,4-FDCA is produced fromfuran-2,4-dicarbaldehyde, and/or -(hydroxymethyl)furoic acidintermediates, wherein: (a) a dehydrogenase, an oxidase, or aperoxigenase catalyzes the conversion of the 4-HMF tofuran-2,4-dicarbaldehyde, and/or 4-(hydroxymethyl)furoic acid; and/or(b) a dehydrogenase, an oxidase, or a peroxigenase catalyzes theconversion of the furan-2,4-dicarbaldehyde from (a) to4-formylfuran-2-carboxylate; and/or (c) a dehydrogenase, an oxidase, ora peroxigenase catalyzes the conversion of the 4-(hydroxymethyl)furoicacid from (a) to 4-formylfuran-2-carboxylate; and/or (d) adehydrogenase, an oxidase, or a peroxigenase catalyzes the conversion ofthe furan-2,4-dicarbaldehyde from (a) to 2-formylfuran-4-carboxylate;and/or (e) a dehydrogenase, an oxidase, or a peroxigenase catalyzes theconversion of the the 4-formylfuran-2-carboxylate from (b) and/or (c) orthe 2-formylfuran-4-carboxylate from (d) to 2,4-FDCA.

In some embodiments, the dehydrogenase from (a), (b), (c), (d) and/or(e) is classified as EC number 1.2.1. or EC number 1.1.1 In someembodiments, the dehydrogenase is an aldehyde dehydrogenase or analcohol dehydrogenase. In some embodiments, the oxidase from (a), (b),(c), (d) and/or (e) is classified as EC number 1.1.3. In someembodiments, the oxidase is 5-(hydroxymethyl)furfural oxidase.

The disclosure provides a 2,4-FDCA produced according to the methods ofthe disclosure.

The disclosure provides a 2,4-FDCA produced according to themicroorganisms of the disclosure.

The disclosure provides a polymer produced from the 2,4-FDCA ofembodiments of the disclosure. In some embodiments, the polymer from2,4-FDCA is formed in a non-biological process.

The disclosure provides a recombinant microorganism capable of producing4-hydroxymethylfurfural (4-HIVIF) from a feedstock comprising anexogenous carbon source, wherein the recombinant microorganism expressesthe following: (a) endogenous and/or exogenous nucleic acid moleculescapable of converting the carbon source to glyceraldehyde 3-phosphate(G3P); (b) at least one endogenous or exogenous nucleic acid moleculeencoding a (5-formylfuran-3-yl)methyl phosphate synthase that catalyzesthe conversion of G3P from (a) to (5-formylfuran-3-yl)methyl phosphate;and (c) at least one endogenous or exogenous nucleic acid moleculeencoding a phosphatase that catalyzes the conversion of(5-formylfuran-3-yl)methyl phosphate from (b) to 4-hydroxymethylfurfural(4-HMF).

In some embodiments, the methyl phosphate synthase from (a) isclassified as EC number 4.2.3.153. In some embodiments, the synthase is(5-formylfuran-3-yl)methyl phosphate synthase. In some embodiments, thephosphatase from (c) is classified as EC number 3.1.3. In someembodiments, the phosphatase is classified as haloacid dehalogenase. Insome embodiments, the phosphatase is endogenous to the host. In someembodiments, the phosphatase enzyme endogenous to the host isoverexpressed.

The disclosure provides a recombinant microorganism capable of producing2,4-furandicarboxylic acid (2,4-FDCA) from a feedstock comprising acarbon source, wherein the recombinant microorganism expresses one ormore of the following: (a) endogenous and/or exogenous nucleic acidmolecules capable of converting glycerol or a monosaccharide toglyceraldehyde 3-phosphate (G3P); (b) at least one endogenous orexogenous nucleic acid molecule encoding a (5-formylfuran-3-yl)methylphosphate synthase that catalyzes the conversion of G3P from (a) to(5-formylfuran-3-yl)methyl phosphate; (c) at least one endogenous orexogenous nucleic acid molecule encoding a phosphatase that catalyzesthe conversion of (5-formylfuran-3-yl)methyl phosphate from (b) to4-hydroxymethylfurfural (4-HMF); (d) at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenases and/or anoxidase that catalyzes independently or in synergy the oxidation of4-HMF from (b) to 2,4 FDCA, directly or through the production ofintermediates furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,4-formylfuran-2-carboxylate, 4-formylfuran-2-carboxylate,2-formylfuran-4-carboxylate.

In some aspects, the disclosure is generally drawn to a method ofproducing 2,4-furandicarboxylic acid (2,4-FDCA) by enzymaticallyconverting glyceraldehyde 3-phosphate (G3P) to 2,4-furandicarboxylicacid (2,4-FDCA) in a recombinant microorganism, by enzymatic catalystsin the absence of microorganisms, the method comprising: (a) providingG3P in the presence of a methyl phosphate synthase or any enzyme able tocatalyzes the conversion of G3P to (5-formylfuran-3-yl)methyl phosphate;(b) providing the (5-formylfuran-3-yl)methyl phosphate from (a) to aphosphatase or any enzyme able to catalyze the conversion of the(5-formylfuran-3-yl)methyl phosphate to 4-hydroxymethylfurfural (4-HMF);(c) providing the 4-HMF from (b) to oxidases, dehydrogenase orperoxigenase able to catalyze independently or in synergy the oxidationof 4-HMF to 2,4 FDCA, directly or through the production ofintermediates furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,4-formylfuran-2-carboxylate, 4-formylfuran-2-carboxylate,2-formylfuran-4-carboxylate.

In this sense, step C could be performed by providing the 4-HMF from (b)to a dehydrogenase or an oxidase or that catalyzes the conversion of the4-HMF to: (i) furan-2,4-dicarbaldehyde, and/or (ii)4-(hydroxymethyl)furoic acid; (d) providing the: (i)furan-2,4-dicarbaldehyde from (c)(i) to a dehydrogenase, an oxidase, ora peroxigenase that catalyzes the conversion of thefuran-2,4-dicarbaldehyde to 4-formylfuran-2-carboxylate; (ii)4-(hydroxymethyl)furoic acid from (c)(ii) to a dehydrogenase, anoxidase, or a peroxigenase that catalyzes the conversion of the4-(hydroxymethyl)furoic acid to 4-formylfuran-2-carboxylate; and/or(iii) furan-2,4-dicarbaldehyde from I(i) to a dehydrogenase, an oxidase,or a peroxigenase that catalyzes the conversion of thefuran-2,4-dicarbaldehyde to 2-formylfuran-4-carboxylate; and (e)providing the 4-formylfuran-2-carboxylate from (d)(i) and/or (d)(ii) orthe 2-formylfuran-4-carboxylate from (d)(iii) to a dehydrogenase or anoxidase that catalyzes the conversion of the 4-formylfuran-2-carboxylatefrom (d)(i) and/or (d)(ii) or the 2-formylfuran-4-carboxylate from(d)(iii) to 2,4-FDCA.

In some aspects, the methyl phosphate synthase from (a) is classified asEC number 4.2.3.153. In some aspects, the synthase is(5-formylfuran-3-yl)methyl phosphate synthase.

In some aspects, the phosphatase from (b) is a Phosphoric monoesterhydrolase classified as EC number 3.1.3. In some aspects, thephosphatase is classified as haloacid dehalogenase (Koonin et al. J.Mol. Biol. 244(1). 1994). In some aspects, the phosphatase of reaction bis endogenous to the host (Offley et al. Curr. Gen. 65. 2019). In someaspects, the phosphatase enzyme endogenous to the host is overexpressed.In some cases a heterologous phosphatase able to perform the desiredreaction is used and is selected from an alkaline phosphatase, acidphosphatase, fructose-bisphosphatase, sugar-phosphatase, orsugar-terminal-phosphatase.

In some aspects, the dehydrogenase from (c) is classified as EC number1.1.1. when oxidizing an alcohol to a carbonyl or EC number 1.2.1. whenoxidizing a carbonyl to an acid. In some aspects, the dehydrogenase isan alcohol dehydrogenase or an aldehyde dehydrogenase.

In some aspects, the oxidase from (c) is classified as EC number 1.1.3.In some aspects, the oxidase is 5-(hydroxymethylfurfural oxidase. Insome aspects the 5-hydroxymethylfurfural oxidase convert the4-hydroxymethylfurfural (4-HMF) into 2,4 FDCA in a three-step reaction.

In some aspects, the disclosure is generally drawn to a recombinantmicroorganism capable of producing 2,4-furandicarboxylic acid (2,4-FDCA)from a feedstock comprising a carbon source, wherein the recombinantmicroorganism expresses the following: (a) endogenous and/or exogenousnucleic acid molecules capable of converting a carbon source toglyceraldehyde 3-phosphate (G3P); (b) at least one endogenous orexogenous nucleic acid molecule encoding a (5-formylfuran-3-yl)methylphosphate synthase that catalyzes the conversion of G3P from (a) to(5-formylfuran-3-yl)methyl phosphate; (c) at least one endogenous orexogenous nucleic acid molecule encoding a phosphatase that catalyzesthe conversion of (5-formylfuran-3-yl)methyl phosphate from (b) to4-hydroxymethylfurfural (4-HMF); (d) at least one endogenous orexogenous nucleic acid molecule encoding a peroxigenase, dehydrogenase,or a oxidase that catalyzes the conversion of 4-HMF from (c) to 2,4 FDCAdirectly or through the production of intermediatesfuran-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,4-formylfuran-2-carboxylate, 4-formylfuran-2-carboxylate,2-formylfuran-4-carboxylate.

In some aspects, 2,4-furandicarboxylic acid (2,4-FDCA) can be producedby providing the 4-HMF from (c) to a dehydrogenase or an oxidase orperoxidase that catalyzes the conversion of the 4-HMF to: (i)furan-2,4-dicarbaldehyde, and/or (ii) 4-(hydroxymethyl)furoic acid; (d)providing the: (i) furan-2,4-dicarbaldehyde from (c)(i) to adehydrogenase, an oxidase, or a peroxigenase that catalyzes theconversion of the furan-2,4-dicarbaldehyde to4-formylfuran-2-carboxylate; (ii) 4-(hydroxymethyl)furoic acid from(c)(ii) to a dehydrogenase, an oxidase, or a peroxigenase that catalyzesthe conversion of the 4-(hydroxymethyl)furoic acid to4-formylfuran-2-carboxylate; and/or (iii) furan-2,4-dicarbaldehyde fromI(i) to a dehydrogenase, an oxidase, or a peroxigenase that catalyzesthe conversion of the furan-2,4-dicarbaldehyde to2-formylfuran-4-carboxylate; and (e) providing the4-formylfuran-2-carboxylate from (d)(i) and/or (d)(ii) or the2-formylfuran-4-carboxylate from (d)(iii) to a dehydrogenase or anoxidase that catalyzes the conversion of the 4-formylfuran-2-carboxylatefrom (d)(i) and/or (d)(ii) or the 2-formylfuran-4-carboxylate from(d)(iii) to 2,4-FDCA.

In some aspects, the host microorganism is genetically modified toimprove G3P availability to the (5-formylfuran-3-yl)methyl phosphatesynthase that catalyzes the conversion of G3P to(5-formylfuran-3-yl)methyl phosphate. Different metabolic engineeringstrategies can be performed to achieve varied levels of gene expressionthrough modification of regulation of transcription (Alper et al. PNAS102(36). 2005), mRNA stability and translation (Ferreira et al. PNAS110(28). 2013) (Salis et al. Nat. Biotech. 27. 2009), protein stability(Cameron et al. Nat. Biotech. 32. 2014) or genes substitution for a lessor more efficient orthologue.

In some aspects, the carbon source comprises a hexose, a pentose,glycerol, CO2, sucroses and/or combinations thereof.

In some aspects, the methyl phosphate synthase from (b) is classified asEC number 4.2.3.153. In some aspects, the synthase is(5-formylfuran-3-yl)methyl phosphate synthase (Table 1).

In some aspects, the phosphatase from (c) is a Phosphoric monoesterhydrolases classified as EC number 3.1.3 In some aspects, thephosphatase is classified as haloacid dehalogenase (Koonin et al. J.Mol. Biol. 244(1). 1994). In some aspects, the phosphatase of reaction cis endogenous to the host (Offley et al. Curr. Gen. 65. 2019). In someaspects, the phosphatase enzyme endogenous to the host is overexpressed.In some cases, a heterologous phosphatase able to perform the desiredreaction is used and is selected from an alkaline phosphatase, acidphosphatase, fructose-bisphosphatase, sugar-phosphatase, orsugar-terminal-phosphatase.

In some aspects, the oxidase from (d) is classified as EC number 1.1.3.In some aspects, the oxidase is 5-hydroxymethylfurfural oxidase. In someaspects the 5-hydroxymethylfurfural oxidase convert the4-hydroxymethylfurfural (4-HMF) into 2,4 FDCA in a three-step reaction.

In some aspects, the dehydrogenase from (d) is classified as EC number1.1.1. when oxidizing an alcohol to a carbonyl or EC number 1.2.1. whenoxidizing an carbonyl to acid. In some aspects, the dehydrogenase is analcohol dehydrogenase or an aldehyde dehydrogenase.

In some aspects, the dehydrogenase from (e) is classified as EC number1.2.1. or EC number 1.1.1 In some aspects, the dehydrogenase is analdehyde dehydrogenase or an alcohol dehydrogenase. In some aspects, theoxidase from (e) is classified as EC number 1.1.3. In some aspects, theoxidase is (5-(hydroxymethyl)furfuraloxidase. In some aspects, thedehydrogenase from (f) is classified as EC number 1.2.1. In someaspects, the dehydrogenase is an aldehyde dehydrogenase. In someaspects, the oxidase from (f) is classified as EC number 1.1.3. In someaspects, the oxidase is (5-(hydroxymethyl)furfural oxidase.

In some aspects, the one or more recombinant microorganisms are derivedfrom a parental microorganism selected from the group consisting ofClostridium sp., Clostridium ljungdahlii, Clostridium autoethanogenum,Clostridium ragsdalei, Eubacterium limosum, Butyribacteriummethylotrophicum, Moorella thermoacetica, Clostridium aceticum,Acetobacterium woodii, Alkalibaculum bacchii, Clostridium drakei,Clostridium carboxidivorans, Clostridium formicoaceticum, Clostridiumscatologenes, Moorella thermoautotrophica, Acetonema longum, Blautiaproducta, Clostridium glycolicum, Clostridium magnum, Clostridiummayombei, Clostridium methoxybenzovorans, Clostridium acetobutylicum,Clostridium beijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui,Sporomusa ovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum,Sporomusa termitida, Moorella glycerini, Eubacterium aggregans,Treponema azotonutricium, Escherichia coli, Saccharomyces cerevisiae,Pseudomonas putida, Bacillus sp., Corynebacterium sp., Yarrowialipolytica, Scheffersomyces stipitis, and Terrisporobacter glycolicus.

In some aspects, the disclosure is generally drawn to a method ofproducing 2,4-FDCA using a recombinant microorganism of the disclosure,the method comprising cultivating the recombinant microorganism in aculture medium containing a feedstock providing a carbon source untilthe 2,4-FDCA is produced.

In some aspects, the disclosure is generally drawn to a method ofproducing a recombinant microorganism capable of producing 2,4-FDCA froma feedstock comprising a carbon source, the method comprisingintroducing into and/or overexpressing in the recombinant microorganismthe following: (a) endogenous and/or exogenous nucleic acid moleculescapable of converting glycerol or a monosaccharide to glyceraldehyde3-phosphate (G3P); (b) at least one endogenous or exogenous nucleic acidmolecule encoding a (5-formylfuran-3-yl)methyl phosphate synthase thatcatalyzes the conversion of G3P from (a) to (5-formylfuran-3-yl)methylphosphate; (c) at least one endogenous or exogenous nucleic acidmolecule encoding a phosphatase that catalyzes the conversion of(5-formylfuran-3-yl)methyl phosphate from (b) to 4-hydroxymethylfurfural(4-HMF); (d) at least one endogenous or exogenous nucleic acid moleculeencoding a peroxigenase, dehydrogenase, or an oxidase that catalyzes theconversion of 4-HMF from (c) to: (i) furan-2,4-dicarbaldehyde and/or(ii) 4-(hydroxymethyl)furoic acid; (e) at least one endogenous orexogenous nucleic acid molecule encoding a peroxigenase, dehydrogenase,or a oxidase that catalyzes the conversion of: (i)furan-2,4-dicarbaldehyde from (d)(i) to 4-formylfuran-2-carboxylateand/or (ii) 4-(hydroxymethyl)furoic acid from (d)(ii) to4-formylfuran-2-carboxylate; and/or (iii) furan-2,4-dicarbaldehyde from(c)(i) to 2-formylfuran-4-carboxylate; and (f) at least one endogenousor exogenous nucleic acid molecule encoding a peroxigenase,dehydrogenase, or an oxidase that catalyzes the conversion of4-formylfuran-2-carboxylate from (e)(i) and (e)(ii) or2-formylfuran-4-carboxylate from (e)(iii) to 2,4-FDCA.

In some aspects, the carbon source comprises a hexose, a pentose,glycerol, and/or combinations thereof. In some aspects, the methylphosphate synthase from (b) is classified as EC number 4.2.3.153. Insome aspects, the synthase is (5-formylfuran-3-yl)methyl phosphatesynthase. In some aspects, the phosphatase from (c) is a Phosphoricmonoester hydrolase classified as EC number 3.1.3. In some aspects, thephosphatase is classified as haloacid dehalogenase (Koonin et al. J.Mol. Biol. 244(1). 1994). In some aspects, the phosphatase of reaction cis endogenous to the host (Offley et al. Curr. Gen. 65. 2019). In someaspects, the phosphatase enzyme endogenous to the host is overexpressed.In some cases, a heterologous phosphatase able to perform the desiredreaction is used and is selected from an alkaline phosphatase, acidphosphatase, fructose-bisphosphatase, sugar-phosphatase, orsugar-terminal-phosphatase.

In some aspects, the dehydrogenase from (d) is classified as EC number1.1.1. when oxidizing an alcohol to a carbonyl or EC number 1.2.1. whenoxidizing a carbonyl to an acid. In some aspects, the dehydrogenase isan alcohol dehydrogenase or an aldehyde dehydrogenase.

In some aspects, the oxidase from (d) is classified as EC number 1.1.3.In some aspects, the oxidase is 5-(hydroxymethylfurfural oxidase. Insome aspects the 5-hydroxymethylfurfural oxidase convert the4-hydroxymethylfurfural (4-HMF) into 2,4 FDCA in a three-step reaction.

In some aspects, the oxidase from (e) is classified as EC number 1.1.3.In some aspects, the oxidase is (5-(hydroxymethyl)furfural oxidase. Insome aspects, the dehydrogenase from (f) is classified as EC number1.2.1. In some aspects, the dehydrogenase is aldehyde dehydrogenase. Insome aspects, the oxidase from (f) is classified as EC number 1.1.3. Insome aspects, the oxidase is (5-(hydroxymethyl)furfural oxidase.

In some aspects, the disclosure is drawn to a method of producing apolymer from 2,4-FDCA produced by the microorganism wherein the 2,4-FDCAand a diol are catalytically polymerized in a non-biological process. Insome aspects the 2,4-FDCA is part of a plasticizer agent composition andwhere the plasticizer agent is part of a plasticized polymercomposition.

In some aspects, the disclosure is generally drawn to a recombinantmicroorganism capable of producing 4-hydroxymethylfurfural (4-HMF) froma feedstock comprising an exogenous carbon source, wherein therecombinant microorganism expresses the following: (a) endogenous and/orexogenous nucleic acid molecules capable of converting the carbon sourceto glyceraldehyde 3-phosphate (G3P); (b) at least one endogenous orexogenous nucleic acid molecule encoding a (5-formylfuran-3-yl)methylphosphate synthase that catalyzes the conversion of G3P from (a) to(5-formylfuran-3-yl)methyl phosphate; and (c) at least one endogenous orexogenous nucleic acid molecule encoding a phosphatase that catalyzesthe conversion of (5-formylfuran-3-yl)methyl phosphate from (b) to4-hydroxymethylfurfural (4-HMF).

In some aspects, the phosphatase is classified as haloacid dehalogenase(Koonin et al. J. Mol. Biol. 244(1). 1994). In some aspects, thephosphatase of reaction b is endogenous to the host (Offley et al. Curr.Gen. 65. 2019). In some aspects, the phosphatase enzyme endogenous tothe host is overexpressed. In some cases, a heterologous phosphataseable to perform the desired reaction is used and is selected from analkaline phosphatase, acid phosphatase, fructose-bisphosphatase,sugar-phosphatase, or sugar-terminal-phosphatase.

In some aspects, the disclosure is generally drawn to a recombinantmicroorganism capable of producing 2,4-furandicarboxylic acid (2,4-FDCA)from a feedstock comprising a carbon source, wherein the recombinantmicroorganism expresses one or more of the following: (a) endogenousand/or exogenous nucleic acid molecules capable of converting glycerolor a monosaccharide to glyceraldehyde 3-phosphate (G3P); (b) at leastone endogenous or exogenous nucleic acid molecule encoding a(5-formylfuran-3-yl)methyl phosphate synthase that catalyzes theconversion of G3P from (a) to (5-formylfuran-3-yl)methyl phosphate; (c)at least one endogenous or exogenous nucleic acid molecule encoding aphosphatase that catalyzes the conversion of (5-formylfuran-3-yl)methylphosphate from (b) to 4-hydroxymethylfurfural (4-HMF); (d) thatcatalyzes the conversion of 4-HMF from (c) to 2,4 FDCA directly orthrough the production of intermediates furan-2,4-dicarbaldehyde,4-(hydroxymethyl)furoic acid, 4-formylfuran-2-carboxylate,4-formylfuran-2-carboxylate, 2-formylfuran-4-carboxylate.

In some aspects, 2,4-furandicarboxylic acid (2,4-FDCA) can be producedby providing the 4-HMF from (c) to at least one endogenous or exogenousnucleic acid molecule encoding a peroxigenase, dehydrogenase, or anoxidase that catalyzes the conversion of 4-HMF from (c) to: (i)furan-2,4-dicarbaldehyde and/or (ii) 4-(hydroxymethyl)furoic acid; (e)at least one endogenous or exogenous nucleic acid molecule encoding aperoxigenase, dehydrogenase, or an oxidase that catalyzes the conversionof: (i) furan-2,4-dicarbaldehyde from (d)(i) to4-formylfuran-2-carboxylate and/or (ii) 4-(hydroxymethyl)furoic acidfrom (d)(ii) to 4-formylfuran-2-carboxylate; and (f) at least oneendogenous or exogenous nucleic acid molecule encoding a peroxigenase,dehydrogenase, or an oxidase that catalyzes the conversion of4-formylfuran-2-carboxylate from (e) to 2,4-FDCA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic overview of the biosynthetic pathway utilized byrecombinant microorganisms of the disclosure for the novel conversion ofG3P to 4-HMF. The numbers below the enzymatic reaction rows indicate the3-digit EC number for the corresponding enzymes.

FIG. 2 is a schematic overview of the biosynthetic production ofproducts contemplated, utilizing 4-HMF as a substrate. The productsinclude, but are not limited to, 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and 2,4-FDCA.The numbers near the enzymatic reaction rows indicate the 3-digit ECnumber for the corresponding enzymes.

FIG. 3 is a schematic overview of possible biosynthetic pathways for theconversion of a carbon source (in this case glucose or glycerol) to G3P.

FIG. 4 is an illustrative SDS-PAGE image of expressed and purified5-formylfuran-3-yl)methyl phosphate synthase candidate, MfnB1.

FIG. 5 is a representative UV spectra showing a negative control sample(grey) and methyl phosphate synthase reaction (black) showing(5-formylfuran-3-yl)methyl phosphate produced from G3P.

FIG. 6 is a representative UV spectra showing (5-formylfuran-3-yl)methylphosphate production from G3P by methyl phosphate synthases at t₀ (Upperpanel) and t_(2h) (Lower panel).

FIG. 7 is a representative UV spectra showing 4-HMF production from(5-formylfuran-3-yl)methyl phosphate by phosphatase (Upper Panel), E.coli lysates (Middle Panel), and yeast lysates (Lower Panel).

FIG. 8 is an illustrative SDS-PAGE image of the expressed the 4-HMFoxidase candidate, HmfH1, in purified form (PE), soluble phase beforepurification (SP), in the insoluble phase (IP), and the flow through(FT) after purification.

FIG. 9 is a representative UV spectra showing 2,4 FDCA production from4-HMF by 4-HMF oxidase candidates HmfH1 (Upper Panel), HmfH6 (MiddlePanel), and HmfH7 (Lower Panel) after 16 hours incubation. Reactionintermediates 4-formylfuran-2-carboxylate (2,4-FFCA) andfuran-2,4-dicarbaldehyde (2,4-DFF) were also identified and quantified.The chromatographic separation was performed by HPLC-DAD.

FIG. 10 is a representative GC-MS chromatogram (Upper Panel) and massspectrum (Lower Panel) showing identification of 2,4-FDCA produced from4-HMF with hMFh7.

FIG. 11 is a representation of silylated 2,4-FDCA.

FIG. 12 is a representative plot showing NAD(P)H depletion due to itsoxidation during the reduction of 2,4-HMF to 2,4-furandimethanol by4-HMF dehydrogenase candidates DH1, DH2, or DH6.

FIG. 13 is a representative plot showing NAD(P)H formation due toreduction of the cofactor and oxidation of the 2,4-HMF substrate tofuran-2,4-dicaraldehyde.

FIG. 14 is a representative plot showing NAD(P)H formation due toreduction of the cofactor and oxidation of the 2,4-HMF substrate to4-(hydroxymethyl)furoic acid by aldehyde dehydrogenase candidates, DH8,DH9, DH10, and DH11.

FIG. 15 is a representative chromatogram showing 2,4-FDCA productionfrom 4-HMF by the combination of an aldehyde dehydrogenase (DH8) and analcohol dehydrogenase (DH6). Negative control reaction (Upper Panel)performed without 4-HMF substrate. Reaction with DH8, DH6, and 4-HMFsubstrate (Middle Panel). Negative control reaction (Lower Panel)performed without DH6 and DH8 enzymes.

FIG. 16 is a representative chromatogram showing the 2,4-FDCA productionin vivo from glucose fermentation at t₀ (Upper Panel) and t_(48h) (LowerPanel).

DETAILED DESCRIPTION

Definitions

The following definitions and abbreviations are to be used for theinterpretation of the disclosure.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “an enzyme” includes aplurality of such enzymes and reference to “the microorganism” includesreference to one or more microorganisms, and so forth.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having, “contains,” “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Acomposition, mixture, process, method, article, or apparatus thatcomprises a list of elements is not necessarily limited to only thoseelements but may include other elements not expressly listed or inherentto such composition, mixture, process, method, article, or apparatus.Further, unless expressly stated to the contrary, “or” refers to aninclusive “or” and not to an exclusive “or.”

The terms “about” and “around,” as used herein to modify a numericalvalue, indicate a close range surrounding that explicit value. If “X”were the value, “about X” or “around X” would indicate a value from 0.9Xto 1.1X, or, in some embodiments, a value from 0.95X to 1.05X. Anyreference to “about X” or “around X” specifically indicates at least thevalues X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X,and 1.05X. Thus, “about X” and “around X” are intended to teach andprovide written description support for a claim limitation of, e.g.,“0.98X.”

As used herein, the terms “microbial,” “microbial organism,” and“microorganism” include any organism that exists as a microscopic cellthat is included within the domains of archaea, bacteria or eukarya, thelatter including yeast and filamentous fungi, protozoa, algae, or higherProtista. Therefore, the term is intended to encompass prokaryotic oreukaryotic cells or organisms having a microscopic size and includesbacteria, archaea, and eubacteria of all species as well as eukaryoticmicroorganisms such as yeast and fungi. Also included are cell culturesof any species that can be cultured for the production of a chemical.

As described herein, in some embodiments, the recombinant microorganismsare prokaryotic microorganism. In some embodiments, the prokaryoticmicroorganisms are bacteria. “Bacteria”, or “eubacteria”, refers to adomain of prokaryotic organisms. Bacteria include at least elevendistinct groups as follows: (1) Gram-positive (gram+) bacteria, of whichthere are two major subdivisions: (1) high G+C group (Actinomycetes,Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus,Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas);(2) Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of gram positive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

The term “recombinant microorganism” and “recombinant host cell” areused interchangeably herein and refer to microorganisms that have beengenetically modified to express or to overexpress endogenous enzymes, toexpress heterologous enzymes, such as those included in a vector, in anintegration construct, or which have an alteration in expression of anendogenous gene. By “alteration” it is meant that the expression of thegene, or level of a RNA molecule or equivalent RNA molecules encodingone or more polypeptides or polypeptide subunits, or activity of one ormore polypeptides or polypeptide subunits is up regulated or downregulated, such that expression, level, or activity is greater than orless than that observed in the absence of the alteration. It isunderstood that the terms “recombinant microorganism” and “recombinanthost cell” refer not only to the particular recombinant microorganismbut to the progeny or potential progeny of such a microorganism.

The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein results from transcription andtranslation of the open reading frame sequence. The level of expressionof a desired product in a host cell may be determined on the basis ofeither the amount of corresponding mRNA that is present in the cell, orthe amount of the desired product encoded by the selected sequence. Forexample, mRNA transcribed from a selected sequence can be quantitated byqRT-PCR or by Northern hybridization (see Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press(1989)). Protein encoded by a selected sequence can be quantitated byvarious methods, e.g., by ELISA, by assaying for the biological activityof the protein, or by employing assays that are independent of suchactivity, such as western blotting or radioimmunoassay, using antibodiesthat recognize and bind the protein. See Sambrook et al., 1989, supra.

The term “decreasing” or “reducing” the level of expression of a gene oran enzyme activity refers to the partial or complete suppression of theexpression of a gene or enzyme activity. This suppression of expressionor activity can be either an inhibition of the expression of the gene, adeletion of all or part of the promoter region necessary for the geneexpression, a deletion in the coding region of the gene, or thereplacement of the wild-type promoter by a weaker natural or syntheticpromoter. For example, a gene may be completely deleted and may bereplaced by a selection marker gene that facilitates the identification,isolation and purification of the strains according to the presentdisclosure. Alternatively, endogenous genes may be knocked out ordeleted to favor the new metabolic pathway. In yet another embodiment,the expression of the gene may be decreased or reduced by using a weakpromoter or by introducing certain mutations.

As used herein, the term “non-naturally occurring,” when used inreference to a microorganism organism or enzyme activity of thedisclosure, is intended to mean that the microorganism organism orenzyme has at least one genetic alteration not normally found in anaturally occurring strain of the referenced species, includingwild-type strains of the referenced species. Genetic alterationsinclude, for example, modifications introducing expressible nucleicacids encoding metabolic polypeptides, other nucleic acid additions,nucleic acid deletions and/or other functional disruption of themicroorganism's genetic material. Such modifications include, forexample, coding regions and functional fragments thereof, forheterologous, homologous, or both heterologous and homologouspolypeptides for the referenced species. Additional modificationsinclude, for example, non-coding regulatory regions in which themodifications alter expression of a gene or operon. Exemplarynon-naturally occurring microorganism or enzyme activity includes thehydroxylation activity described above.

The term “exogenous” as used herein with reference to various molecules,e.g., polynucleotides, polypeptides, enzymes, etc., refers to moleculesthat are not normally or naturally found in and/or produced by a givenyeast, bacterium, organism, microorganism, or cell in nature.

On the other hand, the term “endogenous” or “native” as used herein withreference to various molecules, e.g., polynucleotides, polypeptides,enzymes, etc., refers to molecules that are normally or naturally foundin and/or produced by a given yeast, bacterium, organism, microorganism,or cell in nature.

The term “heterologous” as used herein in the context of a modified hostcell refers to various molecules, e.g., polynucleotides, polypeptides,enzymes, etc., wherein at least one of the following is true: (a) themolecule(s) is/are foreign (“exogenous”) to (i.e., not naturally foundin) the host cell; (b) the molecule(s) is/are naturally found in (e.g.,is “endogenous to”) a given host microorganism or host cell but iseither produced in an unnatural location or in an unnatural amount inthe cell; and/or (c) the molecule(s) differ(s) in nucleotide or aminoacid sequence from the endogenous nucleotide or amino acid sequence(s)such that the molecule differing in nucleotide or amino acid sequencefrom the endogenous nucleotide or amino acid as found endogenously isproduced in an unnatural (e.g., greater than naturally found) amount inthe cell.

The term “homolog,” as used herein with respect to an original enzyme orgene of a first family or species, refers to distinct enzymes or genesof a second family or species which are determined by functional,structural, or genomic analyses to be an enzyme or gene of the secondfamily or species which corresponds to the original enzyme or gene ofthe first family or species. Homologs most often have functional,structural, or genomic similarities. Techniques are known by whichhomologs of an enzyme or gene can readily be cloned using genetic probesand PCR. Identity of cloned sequences as homologs can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.Thus, the term “homologous proteins” is intended to mean that the twoproteins have similar amino acid sequences. In certain instances, thehomology between two proteins is indicative of its shared ancestry,related by evolution. The terms “homologous sequences” or “homologs” arethought, believed, or known to be functionally related. A functionalrelationship may be indicated in any one of a number of ways, including,but not limited to: (a) degree of sequence identity and/or (b) the sameor similar biological function. Preferably, both (a) and (b) areindicated. The degree of sequence identity may vary, but in oneembodiment, is at least 50% (when using standard sequence alignmentprograms known in the art), at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least 98.5%, or at least about 99%, or at least 99.5%, or at least99.8%, or at least 99.9%. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus(Scientific and Educational Software, Pennsylvania). Other non-limitingalignment programs include Sequencher (Gene Codes, Ann Arbor, Mich.),AlignX, and Vector NTI (Invitrogen, Carlsbad, Calif.). A similarbiological function may include, but is not limited to: catalyzing thesame or similar enzymatic reaction; having the same or similarselectivity for a substrate or co-factor; having the same or similarstability; having the same or similar tolerance to various fermentationconditions (temperature, pH, etc.); and/or having the same or similartolerance to various metabolic substrates, products, by-products,intermediates, etc. The degree of similarity in biological function mayvary, but in one embodiment, is at least 1%, at least 2%, at least 3%,at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, atleast 9%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at least98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, orat least 99.9%, according to one or more assays known to one skilled inthe art to determine a given biological function.

The term “variant” refers to any polypeptide or enzyme described herein.A variant also encompasses one or more components of a multimer,multimers comprising an individual component, multimers comprisingmultiples of an individual component (e.g., multimers of a referencemolecule), a chemical breakdown product, and a biological breakdownproduct. In particular, non-limiting embodiments, an enzyme may be a“variant” relative to a reference enzyme by virtue of alteration(s) inany part of the polypeptide sequence encoding the reference enzyme. Avariant of a reference enzyme can have enzyme activity of at least 10%,at least 30%, at least 50%, at least 80%, at least 90%, at least 100%,at least 105%, at least 110%, at least 120%, at least 130% or more in astandard assay used to measure enzyme activity of a preparation of thereference enzyme. In some embodiments, a variant may also refer topolypeptides having at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to the full-length, or unprocessed enzymes of thepresent disclosure. In some embodiments, a variant may also refer topolypeptides having at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to the mature, or processed enzymes of the presentdisclosure.

The term “yield potential” or as used herein refers to a yield of aproduct from a biosynthetic pathway. In one embodiment, the yieldpotential may be expressed as a percent by weight of end product perweight of starting compound.

The term “thermodynamic maximum yield” as used herein refers to themaximum yield of a product obtained from fermentation of a givenfeedstock, such as glucose, based on the energetic value of the productcompared to the feedstock. In a normal fermentation, without use ofadditional energy sources such as light, hydrogen gas or methane orelectricity, for instance, the product cannot contain more energy thanthe feedstock. The thermodynamic maximum yield signifies a product yieldat which all energy and mass from the feedstock is converted to theproduct. This yield can be calculated and is independent of a specificpathway. If a specific pathway towards a product has a lower yield thanthe thermodynamic maximum yield, then it loses mass and can most likelybe improved upon or substituted with a more efficient pathway towardsthe product.

The term “redox balance” refers to the overall amount of redox cofactorsin a given set of reactions. When there is a shortage of redoxcofactors, the redox balance is negative and the yield of such pathwaywould not be realistic since there is a need to burn feedstock tofulfill the cofactor demand. When there is a surplus of redox cofactors,the redox balance is said to be positive and the yield of such pathwayis lower than the maximum yield (Dugar et al. “Relative potential ofbiosynthetic pathways for biofuels and bio-based products” Naturebiotechnology 29.12 (2011): 1074). In addition, when the pathwayproduces the same amount of redox cofactors as it consumes, the redoxbalance is zero and one can refer to this pathway as “redox balanced.”Designing metabolic pathways and engineering an organism such that theredox cofactors are balanced or close to being balanced usually resultsin a more efficient, higher yield production of the desired compoundswhen compared to an unbalanced pathway. Redox reactions always occurtogether as two half-reactions happening simultaneously, one being anoxidation reaction and the other a reduction reaction. In redoxprocesses, the reductant transfers electrons to the oxidant. Thus, inthe reaction, the reductant or reducing agent loses electrons and isoxidized, and the oxidant or oxidizing agent gains electrons and isreduced. In one embodiment, the redox reactions take place in abiological system. The term redox state is often used to describe thebalance of NAD+/NADH and NADP+/NADPH of natural or non-natural metabolicpathways in a biological system such as a microbial cell. The redoxstate is reflected in the balance of several sets of metabolites (e.g.,lactate and pyruvate, beta-hydroxybutyrate, and acetoacetate), whoseinterconversion is dependent on these ratios. In one embodiment, anexternal source of hydrogen or electrons, combined or not with the useof hydrogenase enzymes able to convert hydrogen to NAD(P)H, may bebeneficial to increase product yield in metabolic pathways with negativeredox balance, i.e., when there is a shortage in redox cofactors, suchas NAD(P)H.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA(rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA),micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, andprimers. A polynucleotide may comprise one or more modified nucleotides,such as methylated nucleotides and nucleotide analogs. If present,modifications to the nucleotide structure may be imparted before orafter assembly of the polymer. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule which can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantiallycomplementary” as used herein refers to a degree of complementarity thatis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refersto two nucleic acids that hybridize under stringent conditions. Sequenceidentity, such as for the purpose of assessing percent complementarity,may be measured by any suitable alignment algorithm, including but notlimited to the Needleman-Wunsch algorithm (see e.g. the EMBOSS Needlealigner available atwww.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally withdefault settings), the BLAST algorithm (see e.g. the BLAST alignmenttool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally withdefault settings), or the Smith-Waterman algorithm (see e.g. the EMBOSSWater aligner available atwww.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optionally withdefault settings). Optimal alignment may be assessed using any suitableparameters of a chosen algorithm, including default parameters.

The term “biologically pure culture” or “substantially pure culture”refers to a culture of a bacterial species described herein containingno other bacterial species in quantities sufficient to interfere withthe replication of the culture or be detected by normal bacteriologicaltechniques.

As used herein, a “control sequence” refers to an operator, promoter,silencer, or terminator.

As used herein, “introduced” refers to the introduction by means ofmodern biotechnology, and not a naturally occurring introduction.

As used herein, a “constitutive promoter” is a promoter, which is activeunder most conditions and/or during most development stages. There areseveral advantages to using constitutive promoters in expression vectorsused in biotechnology, such as: high level of production of proteinsused to select transgenic cells or organisms; high level of expressionof reporter proteins or scorable markers, allowing easy detection andquantification; high level of production of a transcription factor thatis part of a regulatory transcription system; production of compoundsthat requires ubiquitous activity in the organism; and production ofcompounds that are required during all stages of development.

As used herein, a “non-constitutive promoter” is a promoter which isactive under certain conditions, in certain types of cells, and/orduring certain development stages. For example, inducible promoters, andpromoters under development control are non-constitutive promoters.

As used herein, “inducible” or “repressible” promoter is a promoterwhich is under chemical or environmental factors control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, certain chemicals, the presenceof light, acidic or basic conditions, etc.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of thedisclosure can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

The term “catalytically polymerized” as used herein refers topolymerization process wherein monomers of the disclosure arepolymerized in a non-biological or non-in vivo context.

The term “signal sequence” as used herein refers to an amino acidsequence that targets peptides and polypeptides to cellular locations orto the extracellular environment. Signal sequences are typically at theN-terminal portion of a polypeptide and are typically removedenzymatically. Polypeptides that have their signal sequences arereferred to as being full-length and/or unprocessed. Polypeptides thathave had their signal sequences removed are referred to as being matureand/or processed.

As used herein, “microbial composition” refers to a compositioncomprising one or more microbes of the present disclosure.

As used herein, “carrier,” “acceptable carrier,” “commerciallyacceptable carrier,” or “industrial acceptable carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the microbe can beadministered, stored, or transferred, which does not detrimentallyeffect the microbe.

As used herein, the term “productivity” refers to the total amount ofbioproduct, such as (2,4-FDCA), produced per hour.

As used herein, the term “biosynthesis products” refers to any one ormore of the following products contemplated herein: 4-HMF,2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoicacid, 2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and2,4-FDCA.

Recombinant Microorganisms

In one embodiment, the present disclosure provides a recombinantmicroorganism capable of producing any one or more of the biosyntheticproducts contemplated herein. In one embodiment, a recombinantmicroorganism produces a 4-HMF. In one embodiment, a recombinantmicroorganism produces a 2,4,furandimethanol. In one embodiment, arecombinant microorganism produces a furan-2,4-dicarbaldehyde. In oneembodiment, a recombinant microorganism produces a4-(hydroxymethyl)furoic acid. In one embodiment, a recombinantmicroorganism produces a 2-formylfuran-4-carboxylate. In one embodiment,a recombinant microorganism produces a 4-formylfuran-2-carboxylate. Inone embodiment, a recombinant microorganism produces a 2,4-FDCA.

In one embodiment, a recombinant microorganism produces any six of thebiosynthetic products. In one embodiment, a recombinant microorganismproduces any five of the biosynthetic products. In one embodiment, arecombinant microorganism produces any four of the biosyntheticproducts. In one embodiment, a recombinant microorganism produces anythree of the biosynthetic products.

In one embodiment, the carbon source is converted to glyceraldehyde3-phosphate (G3P). G3P is a common natural intermediary metabolite. Insome embodiments, it can be produced from glucose via the glycolysispathway or from xylose (like from the pentose phosphate pathway but notlimited) or from glycerol. In some embodiments, G3P can be derived fromCO2 capture technologies. In one embodiment, the recombinantmicroorganism capable of producing any one or more of the biosyntheticproducts utilizing a carbon source that comprises a hexose, a pentose,glycerol, or from CO2 capture technologies. In certain embodiments, thecarbon source is glycerol.

In one embodiment, the recombinant microorganism comprises the novelcapacity to convert G3P to any one or more of the biosynthetic productsvia several enzymatically-catalyzed successive steps.

In one embodiment, the host microorganism is genetically modified toimprove G3P availability to the (5-formylfuran-3-yl)methyl phosphatesynthase that catalyzes the conversion of G3P to(5-formylfuran-3-yl)methyl phosphate.

In one embodiment, the recombinant microorganisms are derived from aparental microorganism selected from the group consisting of Clostridiumsp., Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridiumragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum,Moorella thermoacetica, Clostridium aceticum, Acetobacterium woodii,Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans,Clostridium formicoaceticum, Clostridium scatologenes, Moorellathermoautotrophica, Acetonema longum, Blautia producta, Clostridiumglycolicum, Clostridium magnum, Clostridium mayombei, Clostridiummethoxybenzovorans, Clostridium acetobutylicum, Clostridiumbeijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusaovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum, Sporomusatermitida, Moorella glycerini, Eubacterium aggregans, Treponemaazotonutricium, Escherichia coli, Saccharomyces cerevisiae, Pseudomonasputida, Bacillus sp., Corynebacterium sp., Yarrowia lipolytica,Scheffersomyces stipitis, Methylovorus sp., Cupriavidus sp.,Methanocaldococcus sp. and Terrisporobacter glycolicus.

4-HMF

In one embodiment, the present disclosure comprises converting one ormore carbon sources to glyceraldehyde 3-phosphate (G3P); converting G3Pto (5-formylfuran-3-yl)methyl phosphate (Step A); converting(5-formylfuran-3-yl)methyl phosphate to 4-hydroxymethylfurfural (4-HMF)(Step B).

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises an endogenous and/or exogenousnucleic acid molecules capable of converting a carbon source toglyceraldehyde 3-phosphate (G3P). In one embodiment, glycerol isconverted to glycerol-3-phopshate by at least one endogenous orexogenous nucleic acid molecule encoding a glycerol kinase. In oneembodiment, glycerol-3-phosphate is converted to dihydroxyacetonephosphate (DHAP) by at least one endogenous or exogenous nucleic acidmolecule encoding a glycerol-3-phosphate dehydrogenase. In oneembodiment, glycerol is converted to dihydroxyacetone by at least oneendogenous or exogenous nucleic acid molecule encoding a glyceroldehydrogenase. In one embodiment, dihydroxyacetone is converted todihydroxyacetone phosphate (DHAP) by at least one endogenous orexogenous nucleic acid molecule encoding a dihydroxyacetone kinase. Inone embodiment, DHAP is converted to G3P by at least one endogenous orexogenous nucleic acid molecule encoding a triose phosphate isomerase.See Zhang et al. (2010. Applied and Environmental Microbiology,76.8:2397-2401) for exemplary, but non-limiting, glycerol assimilationpathways contemplated herein.

In one embodiment, the recombinant microorganism of any one of theembodiments of disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a (5-formylfuran-3-yl)methylphosphate synthase that catalyzes the conversion of G3P to(5-formylfuran-3-yl)methyl phosphate. In one embodiment, the(5-formylfuran-3-yl)methyl phosphate synthase is classified as EC number4.2.3.153. In some embodiments the EC 4.2.3.153(5-formylfuran-3-yl)methyl phosphate synthase can be derived from thegene mfnB. In some embodiments, mfnB can be derived fromMethanocaldococcus jannaschii. In some embodiments, the(5-formylfuran-3-yl)methyl phosphate synthase can be derived from enzymecandidates listed at Table 1. In some embodiments the(5-formylfuran-3-yl)methyl phosphate synthase is encoded by an aminoacid sequence listed in Table 1. In some embodiments, the(5-formylfuran-3-yl)methyl phosphate synthase is homologous or similarto the enzymes listed at Table 1. In some embodiments, an(5-formylfuran-3-yl)methyl phosphate synthase enzyme is evolved orengineered to improve its catalytic efficiency, markedly kcat.

TABLE 1 (5-formylfuran-3-yl)methyl phosphate synthases enzymes NameOrganism Sequence MfnB 1 MethanocaldococcusMILLVSPIDVEEAKEAIAGGADIIDVKNPKEGSLGANFPWMIKAIREVT jannaschiiPKDLLVSATVGDVPYKPGTISLAAVGAAISGADYIKVGLYGVKNYYQAVELMKNVVRAVKDIDENKIVVAAGYADAYRVGAVEPLIVPKIARDAGCDVAMLDTAIKDGKTLFDFQSKEILAEFVDEAHSYGLKCALAGSIKKEHIPILKEIGTDIVGVRGAACKGGDRNNGRIDRELVKELKELCK (SEQ ID NO: 1) MfnB 2Methanocaldococcus MILLVSPIDVEEAKEAIAGGADIIDVKNPKEGSLGANFPWMIKAIREVTfervens PKELLVSATVGDVPFKPGTISLAAVGAAISGADYIKVGLYGVKNYYEGVELMKNVVRAVKDIDENKIVVAAGYADAHRVGAVEPLIIPKIARDAGCDVAMLDTAVKDGKTLFDFQSKEILEEFVQESHDYGLKCALAGSIKKEHIPILKEIGTDIVGVRGAVCKGGDRNNGRIDRELVRELKELCK (SEQ ID NO: 2) MfnB 3Methanocaldococcus MILLVSPIDVDEAREAIAGGADIIDVKNPKEGSLGANFPWMIKAIREITvulcanius PKELLVSATVGDVPYKPGTVSLASVGAAMSGADYIKVGLYGVKNYYEAVELMKNVVRAVKDVDENKIVVAAGYADAHRVGAVDPLIIPKIARDADCDVAMLDTAIKDGKTLFDFQSKEILEEFVEETHSYGLKCALAGSIKKEHIPILKEIGTDIVGVRGAVCKGGDRNKGRIDRNLVKELKELV (SEQ ID NO: 3) MfnB 4Methanocaldococcus MLLLVSPIDVEEAKEAIEGGADIIDVKNPKEGSLGANFPWVIREVRKITinfernus PKSLLVSATVGDVPYKPGTVSLAALGAGMSGADYIKVGLYGVKNYNQAVELMKSVVKAVKDFDDNKIVVAAGYADAYRVGAVDPLVIPKIARDSGADVAMLDTAIKDGKTLFDFLSKEILEEFVSEVHDYGLKCALAGTIKKDHIPILKEIGTDIVGVRGAACKGGDRNKGRIDRNLVRELKELC (SEQ ID NO: 4) MfnB 5Methanothermococcus MILLVSPKDVNEAIETIKGGADIVDVKNPPEGSLGANFPWIIKEIREITokinawensis PKNLFVSAAIGDVPYKPGIVALAALGAAMSGADYIKVGLYGIKSYNEAVDLMEKVVKAVKGVDENKIVVAAGYADAHRVGAVEPLIVPKIARDAGCDVAMLDTAVKDGKTLFDHLNEKILAEFVEETHSYGLKCALAGSIKKEEIPILKDINCDIVGVRGAACTKGDRNNGTIKSELVKELSKLCK (SEQ ID NO: 5) MfnB 6Methanococcales MRILISPKDIEEAKEAIEGGADIIDVKNPLEGSLGANFPWVIREIRNITarchaeon HHB PKDRLVSATVGDVPYKPGIVALAAVGAAISGADYIKVGLYGIKSYREAVDVMNKVVKAVKEIDENKIVVAAGYADAYRVGAVDPLIIPKVARDSGCDVAMLDTAVKDGKRLFDHLNRELISEFVEEVHNYGLECALAGSIRKEDIPVLKEIGCDIVGIRGAACTKGDRNNGKIKKELVEELVKLCKNGDK (SEQ ID NO: 6) MfnB 7Methanobrevibacter MLLLISPINHEEALESIKGGADIVDVKNPKEGSLGANFPWVIRDIREITsmithii PEDKLVSATLGDVPYKPGIVSLAAMGAHVSGADYIKVGLYGTKDYDEAVEVMENVAKTIKDVDNDTIVVAAGYADAHRVGAVDPMEIPKVAKDAGCDLAMLDTAVKDGHTLFDYLSIEDLEKFVNEAHSYGLKTALAGSVKKEQLKPLNDIGCDVVGIRGAACVGGDRNTGKIHHSAVAELKELCDSF (SEQ ID NO: 7) MfnB 8Methanobacterium MLLLISPINTQEAREAIDGGADIVDVKNPKEGSLGANFPWVIRNIREITsp. PtaB.Bin024 PKNMKVSATLGDVPYKPGIVALAAAGAIVSGADYIKVGLYGTTNYSEALEVMENVVKIVDEENSDAIVVAAGYADAHRVGAVDPMEIPKIAADSGSDLAMVDTAVKDGKILFDFMNEETLSQFTEQTHEYGLKSALAGSVIEEQLPILAELGCDVVGIRGAACIGGDRNSGSIHHEAVARLKQIV (SEQ ID NO: 8) MfnB 9Methanopyrus sp. MRPRLLVSPVNRDEALEAVEGGAHIIDVKNPEEGSLGANFPWVIREIME KOL6VVPEDREVSATVGDVPYKPGTVAQAVLGVAAVGVDYAKVGLYGTKTEEEALEVMRACSRAVREFGYDIRVVAAGYADAHRVDSIDPMSVPEVAAEAECDVAMVDTAVKDGKRLFDFLREEEVGEFVDLAHEHGLEVALAGSLRHEDMPIVRDLGADIVGIRGAACERGDRNRGAIRSHLVRKLAEALA (SEQ ID NO: 9) MfnB 10Candidatus MTMKLLVSPISVEEARIALDGGADIIDVKNPKEGSLGANFPDVIQSVKRArgoarchaeum VITKPMSVAIGDFNYKPGTASLAALGASVAGADYIKIGLFDVQTREQASethanivorans EMTERVTKAVKQYDSKKKVVICGYSDYNRINSISPFELPGIVSDAGADVVMMDTGVKDGRSTLEFLNLEKLESFIGSAHQYGLLAAIAGSLTFEDIEALKEVAPDIIGVRGCVCGGDRNSSIKLELVRELKERIHH (SEQ ID NO: 10) MfnB 11Methanobacterium MLLLISPINTEEAREAIEGGADIVDVKNPKEGSLGANFPWVIKSISELTcongolense PEGMYVSATLGDVPYKPGIVSLAAAGAVVSGADYIKVGLYGTKNYEEALEVMKNVVKTVKDFNEDAVVVAAGYADAHRVGAVDPMEIPRVAADAGADLAMVDTAVKDGKILFDFMDEDTLIKFNNTIHDYGLKSALAGSVKKEQLEMLYNIGCDVVGIRGAACVGGDRNTGKIHRSAVGELKKMIENF (SEQ ID NO: 11) MfnB12Methanobrevibacter MLLLISPINNEEALESIEGGADIVDVKNPKEGSLGANFPWVISEIRKMTarboriphilus PDDMLVSATLGDVPYKPGIVSLAAMGALTSGADYIKVGLYGISNYDEALEVMINVVKIVKSNNPNATVVASGYGDAHRVGAVSPWDIPKVAKESGSDLAMLDTAVKDGKTLFDYLNIDDLKKFVEETHSYGLKSALAGSVKKEQLKPLYDIGCDVVGVRGAACTGGDRNNGKISRTAVAELKELVNSFD (SEQ ID NO: 12) MfnB 13Methanococcus MILLVSPKDVAEAHEAIEGGADIIDVKNPPEGSLGANFPWVIKETREATmarrpaludis PEGMLVSAAIGDVPYKPGIVTLAALGAAISGADYIKVGLYGIRSYQEALDVMKNVTKAVKDSGENKIVVAAGYADAYRVGGVDPLIIPRVARDAGCDVAMLDTAVKDGKTLFDHMSIELLKEFVEETHKYGMKCALAGSIKIEEIPMLKEINCDIVGVRGAACTKGDRNEGRIQKDLVKEIVKVCRQ (SEQ ID NO: 13) MfnB 14Methanococcus MILLVSPKDVAEAYEAINGGADIIDVKNPPEGSLGANFPWVIKEIRSATvannielii PNGMLVSAAIGDVHYKPGIVTLAALGATISGADYIKIGLYGIRSYQEAVDVMKNVSNAVKSEDPKKIVVAAGYADAYRVGAVDPLIIPKIARDSGCDVAMLDTAVKDGKTLFDHLSIDLLKEFVEETHKYGMKCALAGSIKKEEIPMLKEIGCDIVGIRGAACTKGDRNEGKIQKDLVKEIVKICKE (SEQ ID NO: 14) MfnB 15Methanosarcina MKLLVSPINREEAIIASLGGADIVDVKNPKEGSLGANFPWVIRDVKEVVacetivorans NGRQPISATIGDFNYKPGTASLAALGAAVAGADYIKVGLYDIQTEAQALELLTKITLAVKDYDPSKKVVASGYSDYKRINSISPLLLPAVAAEAGVDVVMVDTGIKDGKSTFEFMDEQELKEFTDLAHEHGLENAIAGSLKFEDLPVLERIGPDIIGVRGMVCGGDRRTAIRQELVEKLVAECQI (SEQ ID NO: 15) MfnB 16Methanosarcina MKLLISPINKEEAIIASRGGADIVDVKNPKEGSLGANFPWVIRDVKGAV barkeriNGRQPISATIGDFNYKPGTASLAAFGAAMAGADYIKVGLYDIQTEDQALELITKITQAVKDYDSTKKVVASGYSDYKRINSISPLLLPSIAAKAGADVVMVDTGIKDGKSTFEFMDEEELKKFTGLAHECGLENAIAGSLKFEDLPVLERIGPDIIGVRGMVCGGDRTNSIRQELVEKLVAECQA (SEQ ID NO: 16) MfnB 17Methylorubrum MSDIVSISSARPRLLVSVRGPDEALTALRAGADLIDAKDPERGALGALPextorquens PETVRAIVAGVGGRAVISAVAGDGIGREIAAAIATIAATGVDFIKIAVGGADDAALAEAAAQAPGRVIGVLFAEDDVAEDGPARLAAAGFVGAMIDTRGKSGTILTSLMAAPQLAAFVAGCRTHGLMSGLAGSLGLGDIPVLARLDPDYLGFRGGLCRASDRRQALDGARVAQAVEAMRAGPRADAA (SEQ ID NO: 17) MfnB 18Methylobacterium MTRPEPHLSVRAAPRLLVSVRDAAEAEVARAAGADLVDAKDPARGALGA sp.LDPALVRAMVARIGDRATTSAVAGEPREAGDLVAKVAAMAATGVDYVKVALPPGLRSGRDGLREAADAARGRLIAVLFAEDGLDLAVLPTLADAGFVGAMIDINTKDGRRLTDRIAVPALSAFTAACRAEGLVSGLAGSLALADIPALSDLGAGYLGFRGGLCRGGDRRGDLDPARIAEAARLLRAGGRRDAA (SEQ ID NO: 18) MfnB 19Methanosarcina MKLLVSPINSEEAIIASIGGADIVDVKNPKEGSLGANFPWVIREVKAVV mazeiNGRQPISATIGDFNYKPGTAALAALGAAVAGADYIKVGLYDIQTESQALELLTKITRAVKDYNPLKKVVASGYSDYKRINSISPLLLPAVAAEAGVDVVMVDTGVKDGKSTFEFMDEKELKEFTDLAHSYGLENAIAGSLKFEDIPLLERIGPDIIGVRGMVCGGDRSTSIRQELVEKLVAECQA (SEQ ID NO: 19) MfnB 20Methyloversatilis MIRMLASVRNLDEARIVLEAGVDLIDLKQPADGALGALPAEVIREVVDFuniversalis VAGRILTSATAGNVEPDAQAVQSAMARIAATGVDYVKAGLFPGNWQQGGRDYAAVRACLRGLTPLAGARRIAVMFADLSPPLALVDAVADAGFDGVMVDTALKIGHSLPDVASTEWLSGFVERARARGLLCGLAGSLRVIHIPALAQRCPDYLGFRGALCAGQARAQALDARAVLAVREALEKVQRLAA (SEQ ID NO: 20) MfnB 21Nitrosococcus MSCWLASVRNLEEISCLLAEGPDIIDFKEPKEGVLGALPLETVREAVAL watsoniiIGRRCQTSAAIGDFPVDSPQIYQRVLEMAATGVDYVKIGLPSNIQQAAACLLSLRPLADQGVSMVGVIFADKRPDFSWTYLIGQAGFKGIMLDTAIKDDFGLLSHLSLSELNNFVKLARSVRLISGLAGSLSIQDIPKLLPLRADYLGFRSALCVAARNRCSRLDPKAVLLIKQAMRENLRIFEI (SEQ ID NO: 21) MfnB 22Streptomyces MKEPTLLLLISPDSVEEALDCAKAAEHLDIVDVKKPDEGSLGANYPWVIcattleya NRRL REIRDAIPADKPVSATVGDVPYKPGTVAQAALGAVVSGATYIKVGLYGC 8057TTPDQVVEVMRGVVRAVKDHRPDALVVASGYADAHRIGCVNPLAIPGVAQRSGCDAAMLDTAVKDGTRLFDHVPPDVCGEFVRLAHEGGLLAALAGSVKAEDLGALTRIGTDIVGVRGAVCEGGDRNAGRIQPHLVAAFRAEMDRHAREHAAVVTPTG (SEQ ID NO: 22) MfnB 23 StreptomycesMLLLISPDGVDEALDCAKAAEHLDIVDVKKPDEGSLGANYPWVIREIRA coelicolorAVPADKPVSATVGDVPYKPGTVAQAALGAAVSGATYIKVGLYGCATPEQAVEVMRGVVRAVKDHRADAFVVASGYADAHRIGCVNPLSLPDIARRSGSDAAMLDTAIKDGTRLFDHVPPDVCAEFVRRAHDCGLLAALAGSVRSGDLGELARIQTDIVGVRGAVCEGGDRTTGRIRPHLVAAFRAEMDRHVREHAA AAAQS (SEQ ID NO: 23)MfnB 24 Streptomyces MLLISPDSVEEALECAKAAQHLDIVDVKKPDEGSLGANHPWVIRAVRDAEFF88969 VPADKPVSATVGDVPYKPGIVAQAALGATVSGATYIKVGLYGCTTPDQAVEVMRGVVRAVKDFRPDALVVASGYADAHRIGCVNPLALPDIARRSGSDGAMLDTAVKDGTRLFDHTPPQVCAEFVRLAHEAGLLAALAGSVKAGDLAELAGMGTDIVGVRGAVCEGGDRNAGRIRPELVAAFRAEMDRCVQQHGGQGAAVAAAS (SEQ ID NO: 24) MfnB 25 StreptomycesMLLLISPDGVEEALACATAAEHLDIVDVKKPDEGSLGANFPWVIREIRA griseusAVPADKPVSATVGDVPYKPGTVAQAALGAAVSGATYIKVGLYGCATPDQAIDVMRGVVRAVKDFRADAFVVASGYADAHRIGCVNPLALPDIARRAGADAAMLDTAIKDGTRLFDHVPPEGCAEFVRLAHEAGLLAALAGSVKAADLATLTRIGTDIVGVRGAVCEGGDRDAGRIQPRLVAAFRAEMDRHARAFAA APAAS (SEQ ID NO: 25)MfnB 26 Streptomyces sp.MLLLISPDGVEEALDCAKAAEHLDIVDVKKPDEGSLGANFPWVIREIRE DH-12AVPADKPVSATVGDVPYKPGTVAQAALGAVVSGATYIKVGLYGCTTPDQGIDVMRAVVRAVKEHNPDALVVASGYADAHRIGCVNPLAVPDIAARSGADAAMLDTAVKDGTRLFDHVPPDVCAEFVRLAHASGRLAALAGSVRQDDLGELTRIGTDIVGVRGAVCEGGDRNAGRIQPHLVAAFRAEMDRYDRERTA GLPAAR (SEQ ID NO: 26)MfnB 27 Streptomyces MLLLISPDSVEEALDCVKAAEHLDIVDVKKPDEGSLGANFPWVIREIRDvenezuelae AVPADKPVSATVGDVPYKPGIVAQAALGAVVSGATYIKVGLYGCTIPEQGIEVMRAVVRAVKDHRPDALVVASGYADAHRVGCVNPLAVPDIAARSGADAAMLDTAIKDGTRLFDHVPPDACAEFVRRAHASGLLAALAGSITQADLGPLIRMGIDIVGVRGAVCAGGDRNAGRIQPHLITAFRAEMDRQGREYAV GIPAAN (SEQ ID NO: 27)

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a phosphatase or a kinase thatcatalyzes the conversion of (5-formylfuran-3-yl)methyl phosphate to(4-HMF). In one embodiment, the phosphatase is classified as haloaciddehalogenase (Koonin et al. J. Mol. Biol. 244(1). 1994). In someaspects, the phosphatase of reaction b is endogenous to the host (Offleyet al. Curr. Gen. 65. 2019). In some aspects, the phosphatase enzymeendogenous to the host is overexpressed. In some cases a heterologousphosphatase able to perform the desired reaction is used and is selectedfrom an alkaline phosphatase, acid phosphatase, fructose-bisphosphatase,sugar-phosphatase, or sugar-terminal-phosphatase. In some embodiments,the phosphatase can be derived from enzyme candidates listed at Table 2.In some embodiments, the phosphatase is homologous or similar to theenzymes listed at Table 2. In some embodiments the 4-HMF phosphataseenzyme is encoded by an amino acid sequence listed in Table 2. In someembodiments, a phosphatase enzyme is evolved or engineered to improveits catalytic efficiency and or specificity for the conversion of(5-formylfuran-3-yl)methyl phosphate to (4-HMF).

TABLE 2 4-HMF phosphatase enzymes Name Organism Sequence PH1Streptomyces MMPEPPRERRTAANRSPAIRPIAFFDVDETLITAKSMLDFARQAPHSLRcoelicolor DDITAQASGQRHSADADLTAMRRRGASRVEMNRVYYRRYAGVSLARLQEAGRDWYHAYRTRPDGYVRAGLAALARHRRAGHTIVLISGSARPLLTPLAQDLGADRILCTEQFADAQGVLTGEVNRPMIGEAKAEAVTEVMAKRGVVPADCFAYGDHESDFGMLQAVGNPVVVGTDLVLVRHAQGSNWPVLPADAGPRCACARRPGPLGHDDPSAIG (SEQ ID NO: 28) PH2 Streptomyces sp.MMPEPPRERRTAANRSPAIRPIAFFDVDETLITAKSMLDFARQAPHSLR E5N91DDITAQASGQRHSADADLTAMRRRGASRVEMNRVYYRRYAGVSLARLQEAGRDWYHAYRTRPDGYVRAGLAALARHRRAGHTIVLISGSARPLLTPLAQDLGADRILCTEQFADAQGVLTGEVDRPMIGEAKAEAVTEVMAKRGVVSADCFAYGDHESDFGMLQAVGNPVVVGTDLVLVRHAQASNWPVLPADAGPRCACARRPGPLGHDDPSAIG (SEQ ID NO: 29) PH3 Streptomyces sp.MSALRHERRAAVSRPVVIRHIAFFDVDETLITAKSLLDFAQRVPHGLWE NRRL S-31DETGQPIERLRSGEIDLAALQRSGASRAEMNRAYYRRYAGVPLERLQKAGRDWYHAYRMRPDGYITAGLAALARHRRAGHMIVLISGSARPLLTPLSEDLGADRILCTEQLDDAQGVLTGEVAHPMVGEAKAEAVTEVMAQLRVPTTDCFAYGDHGSDLDMLQAVGSPVVVGTDPVLARHAQASNWPMLPADAGPRIARAQHHDTSAQYGPQVIALASGRGAAPRRQERW (SEQ ID NO: 30) PH4 StreptomycesMNASIAPAAFFDVDETLVNTKSMEHFLRFWMARQGDDGSGHEAVMAGVR aureusRAAASGVHRSEINRAYYRRFAGVPYAALLEAGRDWWQEYRRGSDAVVVPAWAAATRHRKAGHLVVLVSGSFRGCLEPLAQDLGAHRILCSEPLVDTDGRLTGEVVRPMIGSVKADAVRETVAELGLTAADCSCYGDHSSDLDMLGAVGNPVVVGGDRVLLEHAQRLDWPVLPATPGHLPSPDASPARLLTAAERR (SEQ ID NO: 31) PH5Saccharothrix MSTPPAVAFFDVDETVIKVKSMFEFLRHWMTAQGDDGSAYESFMAGVRE syringaeLADAGVPRAEVNRHYYRRYAGASAADVRAAGEDWYASYRRRPDGFLTATVAAVAAHRAAGNRVVLVSGSFLPVLGPLMADVGADEALCGDPEVGPDGRYTGAIAVPMIGENKTAAVRARMAELGVDPADCYAYGDHQSDLGMLEAVGNPVVVGEDPVLVGKAEAGGWRRLPATTGPLGVPPRVLSVVE (SEQ ID NO: 32) PH6Rhodococcus sp. MTHTGSRPVQVAFFDVDETLITVKSMFAFLEHWLRERGDDGSEYSRLLAMTM3W5.2 ALRRASDEGAPREEVNRSYYRTFRGVPLVELEESGRRWYREFESTAAPYYADTLAALRDHRDAGAAIVLLSGSFAPALGPIGEAVCADRIVASRPVTDGHGVLTGEVERPMIGKAKAEAVTSVLEELGIDTGNSYGYGDHDSDLAFLEAVGHPGLRGSDPVLRAHAARNRWRVLGSATTGLAGAVPLLAATSTGQR GLR (SEQ ID NO: 33)PH7 Rhodococcus sp. MIGTGPRPGQVAFFDVDETLITVKSMFAFLEHWLWERGDDGSEYARLLGUNC363MFTsu5.1 ALRRQSDEGAPREEVNRSYYRTFRGVPLVELEESGRRWYREFESTNAPYYAATLAALHAHREAGAAIVLLSGSFAPALVPIGEAVGADRIVASRPVTDQGGVLIGEVERPMIGQAKAEAVISVQAELGVDAENSYGYGDHESDLAFLEAVGHPGLRGDDQVLLARAARDRWRSLGSETTGLAGAGPLAGSASAGLA QRGIL (SEQ ID NO: 34)PH8 Buttiauxella MHTSAAFFDVDETLITVKSMFDFYDFWCRENNEYDKLQRYMTDFRSAVKwarmboldiae NGTPREQLNREYYRQFAGVNYKDLEEAGKNWERGKKLDSELFISSAVAALKKHQANNMFIVFISGSMHPVLSPVANYLGVIDILCIPLELTGEGIITGEIGTPQTIGIGKKEALINFCSQKKISAADCYAYGDDLSDIPMLESVGYPVCVGKYTELARHAINQRWPVI (SEQ ID NO: 35) PH9 ChaniaMRQTAFYDVDDTLINIKSMFDFFQFWASENGLISQQEQFDSQFSVLARK multitudinisentensMSSREELNRAYYRFFKGVPLLKIEQCAERWFKNSFSNTEIFISYTLKSILAHRVLGHNIVLVSGSMTPLLKPIAQLLGITDILCIKLATDQSGVVTGEILETQTIGEGKAIVIRQYALENDINLSACFAYGDDVSDIPMLACVGHPICIGEGTALSHYASNNNWPIVRVE (SEQ ID NO: 36) PH10 MethylosinusMMEHRSFAFFDVDETLISIKSMFDFFPFWCKWIGAAPEAYSRFETEIAS sporiumAIARHATREELNRLYYRSFRGAQLPVLEAAGAAWFLQRFGRSPPYRKHVVARLEKHRQEGVVPVLVSGSMRPLLRPIARELQAEHCLCTQLVVDESGRLIGEIGSPQTIGEGKAEAIRAFLREQGGRPADCLAYGDDISDLAMLELVGAPVVVGAQPDLLSICRQRDWPYLPL (SEQ ID NO: 37) PH11 KlebsiellaMQQAAAFFDVDETLINIKSMFDFFDFWCKENNEPIKLHKYMANFQSEVK oxytocaKGIPREHLNREYYRQFAGISYKALEEAGEKWERFKLNSELFIGSAVSALKKHQAENMDIVFISGSMLPVLSPVARYLGVKDILCIPLKFTAAGEMTGEIGYPQTIGDGKKDALLQFCEQRNINPSDCYAYGDDLSDIPMLASIGHPVCVGKHSALARHAITHRWQVI (SEQ ID NO: 38) PH12 SerratiaMISAAAFFDVDETLIKMKSMEHEYHYWSNVRGNQKAYEEFIKREQQAVAEGVPREVLNRMYYRQFSGIDIDDVYQVAEDWFHKYLHEKEAYIASAVDRFQRHKISGHLTVFISGSMLPLLKPLGQRLGADAILCTQLLLDAKGKLIGEIGEPQTIGQGKQRALLSFSQSHHIDLAKSFAYGDDLSDIPMLAATGNPVCVGEHSNLAEYARRNNWNMLAENATN (SEQ ID NO: 39) PH13 SaccharomycesMKTIIISDFDETITRVDTICTIAKLPYLLNPRLKPEWGHFIKTYMDGYH cerevisiae ycr015cKYKYNGIRSLPLLSSGVPIIISQSNENKLFADELKYQNHNRVVELNSVNEITKQQIFKSISLDQMKTFARDQNHEDCLLRDGFKTFCSSVVKNFESDFYVLSINWSKEFIHEVIGDRRLKNSHIFCNDLKKVSDKCSQSYNGEFDCRLLIGSDKVKILGEILDKIDSGCNKEGNSCSYWYIGDSEIDLLSILHPSTNGVLLINPQENPSKFIKITEKIIGIPKDKISSFEADNGPAWLQFCEKEGGKGAYLVKSWDSLKDLIMQVTKM (SEQ ID NO: 40) PH14 SaccharomycesMTAQQGVPIKITNKEIAQEFLDKYDTFLFDCDGVLWLGSQALPYTLEIL cereyislae yd1236wNLLKQLGKQLIFVTNNSTKSRLAYTKKFASFGIDVKEEQIFTSGYASAVYIRDFLKLQPGKDKVWVFGESGIGEELKLMGYESLGGADSRLDTPFDAAKSPFLVNGLDKDVSCVIAGLDTKVNYHRLAVTLQYLQKDSVHFVGINVDSTFPQKGYTFPGAGSMIESLAFSSNRRPSYCGKPNQNMLNSIISAFNLDRSKCCMVGDRLNTDMKFGVEGGLGGILLVLSGIETEERALKISHDYPRPKFYIDKLGDIYILTNNEL (SEQ ID NO: 41) PH15 SaccharomycesMTIAKDYRTIYRNQIKKQIRLNQEHLQSLTHLGSQINFEVDPPKLPDPD cerevisiae yd1236wRARKVEFFDIDNTLYRKSTKVQLLMQQSLSNEFKYELGEDDDEAERLIESYYQEYGLSVKGLIKNKQIDDVLQYNTFIDDSLPLQDYLKPDWKLRELLINLKKKKLGKFDKLWLFINSYKNHAIRCVKILGIADLFDGITYCHYDRPIEEEFICKPDPKFFETAKLQSGLSSFANAWFIDDNESNVRSALSMGMGHVIHLIEDYQYESENIVTKDHKNKQQFSILKDILEIPLIMDVEVYRPSSIAIKEMEELEEEGEAYNWSNQQINVQSS (SEQ ID NO: 42) PH16 SaccharomycesMGLITKPLSLKVNAALFDVDGIIIISQPAIAAFWRDEGKDKPYFDAEHV cerevisiae yer062cIQVSHGWRTFDAIAKFAPDFANEEYVNKLEAEIPVKYGEKSIEVPGAVKLCNALNALPKEKWAVATSGTRDMAQKWFEHLGIRRPKYFITANDVKQGKPHPEPYLKGRNGLGYPINEQDPSKSKVVVFEDAPAGIAAGKAAGCKIIGIATTFDLDFLKEKGCDIIVKNHESIRVGGYNAETDEVEFIFDDYLYAKD DLLKW (SEQ ID NO: 43)PH17 Saccharomyces MSIAEFAYKEKPETLVLFDVDGILTPARLIVSEEVRKTLAKLRNKCCIGcerevisiae yfl045c FVGGSDLSKQLEQLGPNVLDEFDYSFSENGLTAYRLGKELASQSFINWLGEEKYNKLAVFILRYLSEIDLPKRRGTFLEFRNGMINVSPIGRNASTEERNEFERYDKEHQIRAKFVEALKKEFPDYGLIFSIGGQISFDVFPAGWDKTYCLQHVEKDGFKEIHFFGDKTMVGGNDYEIFVDERTIGHSVQSPDDTVKILTELFNL (SEQ ID NO: 44) PH18 SaccharomycesMTVEYTASDLATYQNEVNEQIAKNKAHLESLTHPGSKVTFPIDQDISAT cerevisiae ygl224cPQNPNLKVFFFDIDNCLYKSSTRIHDLMQQSILRFFQTHLKLSPEDAHVLNNSYYKEYGLAIRGLVMFHKVNALEYNRLVDDSLPLQDILKPDIPLRNMLLRLRQSGKIDKLWLFTNAYKNHAIRCLRLLGIADLFDGLTYCDYSRTDILVCKPHVKAFEKAMKESGLARYENAYFIDDSGKNIETGIKLGMKTCIHLVENEVNEILGQTPEGAIVISDILELPHVVSDLF (SEQ ID NO: 45) PH19 SaccharomycesMPQFSVDLCLFDLDGTIVSITTAAESAWKKLCRQHGVDPVELFKHSHGA cerevisiae yhr043cRSQEMMKKFFPKLDNIDNKGVLALEKDMADNYLDTVSLIPGAENLLLSLDVDTETQKKLPERKWAIVISGSPYLAFSWFETILKNVGKPKVFITGFDVKNGKPDPEGYSRARDLLRQDLQLIGKQDLKYVVFEDAPVGIKAGKAMGAITVGITSSYDKSVLFDAGADYVVCDLIQVSVVKNNENGIVIQVNNPLIR D (SEQ ID NO: 46) PH20Saccharomyces MAEFSADLCLFDLDGTIVSTIVAAEKAWTKLCYEYGVDPSELFKHSHGAcerevisiae yhr044c RTQEVLRRFFPKLDDIDNKGVLALEKDIAHSYLDTVSLIPGAENLLLSLDVDTETQKKLPERKWAIVISGSPYLAFSWFETILKNVGKPKVFITGFDVKNGKPDPEGYSRARDLLRQDLQLIGKQDLKYVVFEDAPVGIKAGKAMGAITVGITSSYDKSVLFDAGADYVVCDLIQVSVVKNNENGIVIQVNNPLIR A (SEQ ID NO: 47) PH21Saccharomyces MPLITKPLSLKINAALFDVDGIIIISQPAIAAFWRDFGKDKPYFDAEHVcerevisiae yil053w IHISHGWRTYDAIAKFAPDFADEEYVNKLEGEIPEKYGEHSIEVPGAVKLCNALNALPKEKWAVATSGTRDMAKKWFDILKIKRPEYFITANDVKQGKPHPEPYLKGRNGLGFPINEQDPSKSKVVVFEDAPAGIAAGKAAGCKIVGIATTFDLDFLKEKGCDIIVKNHESIRVGEYNAETDEVELIFDDYLYAKD DLLKW (SEQ ID NO: 48)PH22 Saccharomyces MIGKRFFQTTSKKIAFAFDIDGVLFRGKKPIAGASDALKLLNRNKIPYIcerevisiae ykr070w LLINGGGESERARTEFISSKLDVDVSPLQIIQSHIPYKSLVNKYSRILAVGTPSVRGVAEGYGFQDVVHQTDIVRYNRDIAPFSGLSDEQVMEYSRDIPDLTIKKFDAVLVFNDPHDWAADIQIISDAINSENGMLNTLRNEKSGKPSIPIYFSNQDLLWANPYKLNRFGQGAFRLLVRRLYLELNGEPLQDYTLGKPTKLTYDFAHHVLIDWEKRLSGKIGQSVKQKLPLLGTKPSTSPFHAVFMVGDNPASDIIGAQNYGWNSCLVKIGVYNEGDDLKECKPILIVNDVFDAVTKTLEKYA (SEQ ID NO: 49) PH23 SaccharomycesMVKAVIFTDEDGIVTLEDSNDYLIDTLGEGKEKRLKVFEGVLDDIKSFR cerevisiae ynl010wQGFMEMLESIHTPFPECIKILEKKIRLDPGFKDIFEWAQENDVPVIVVSSGMKPIIKVLLTRLVGQESIHKIDIVSNEVEIDAHDQWKIIYKDESPFGHDKSRSIDAYKKKFESTLKAGEQRPVYFYCGDGVSDLSAAKECDLLFAKRGKDLVTYCKKQNVPFHEFDTFKDILASMKQVLAGEKTVAELMEN (SEQ ID NO: 50) PH24Saccharomyces MTKLQGLQGLKHIKAVVEDMDGILCLPQPWMFPAMRNAIGLEDKSIDILcerevisiae yor131c HFIDTLPTEKEKKEAHDRIELVEAKAMKEMQPQPGLVDIMRYLIKNGISKNICTRNVGAPVETFVKRFIPSELSRFDYIVTREFRPTKPQPDPLLHIASKLNIRPLEMIMVGDSFDDMKSGRSAGCFTVLLKNHVNGHLLLEHKELVDVSVEDLSEIIELIQNMNKESF (SEQ ID NO: 51) PH25 SaccharomycesMSSRYRVEYHLKSHRKDEFIDWVKGLLASPFVLHAVSHEGDYNDDLATT cerevisiae yor155cQRVRSQYADIFKDIEGLIKDKIEFDSRNMSQDEIEDGASSQSLNILGQSRLNLLVPSIGIFFTELPLEQAFLWEDSQRAISARRMVAPSFNDIRHILNTAQIFHFKKQENLHNGKVLRLVTFDGDVTLYEDGGSLVYTNPVIPYILKLLRCGINVGIVTAAGYDEAGTYENRLKGLIVALHDSTDIPVSQKQNLTIMGGESSYLFRYYEDPEEDNFGFRQIDKEEWLLPRMKAWSLEDVEKTLDFAERTLNRLRKRLNLPSEISIIRKVRAVGIVPGERYDEASKRQVPVKLDREQLEEIVLTLQNTLESFAPSRRIQFSCFDGGSDVWCDIGGKDLGVRSLQQFYNPESPIQPSETLHVGDQFAPVGSANDFKARLAGCTLWIASPQETVNYLHRLLETD (SEQ ID NO: 52) PH26 Escherichia coliMSTPRQILAAIFDMDGLLIDSEPLWDRAELDVMASLGVDISRRNELPDT YniCLGLRIDMVVDLWYARQPWNGPSRQEVVERVIARAISLVEETRPLLPGVREAVALCKEQGLLVGLASASPLHMLEKVLTMFDLRDSFDALASAEKLPYSKPHPQVYLDCAAKLGVDPLTCVALEDSVNGMIASKAARMRSIVVPAPEAQNDPRFVLANVKLSSLTELTAKDLLG (SEQ ID NO: 53) PH27 Escherichia coliMRCKGFLFDLDGTLVDSLPAVERAWSNWARRHGLAPEEVLAFIHGKQAI YfbTTSLRHFMAGKSEADIAAEFTRLEHIEATETEGITALPGAIALLSHLNKAGIPWAIVISGSMPVARARHKIAGLPAPEVEVTAERVKRGKPEPDAYLLGAQLLGLAPQECVVVEDAPAGVLSGLAAGCHVIAVNAPADTPRLNEVDLVLHSLEQIIVIKQPNGDVIIQ (SEQ ID NO: 54) PH28 Escherichia coliMSTPRQILAAIFDMDGLLIDSEPLWDRAELDVMASLGVDISRRNELPDT YieHLGLRIDMVVDLWYARQPWNGPSRQEVVERVIARAISLVEETRPLLPGVREAVALCKEQGLLVGLASASPLHMLEKVLTMFDLRDSFDALASAEKLPYSKPHPQVYLDCAAKLGVDPLTCVALEDSVNGMIASKAARMRSIVVPAPEAQNDPRFVLADVKLSSLTELTAKDLLG (SEQ ID NO: 55) PH29 Escherichia coliMLYIFDLGNVIVDIDFNRVLGAWSDLTRIPLASLKKSFHMGEAFHQHER YihXGEISDEAFAEALCHEMALPLSYEQFSHGWQAVFVALRPEVIAIMHKLREQGHRVVVLSNTNRLHTTFWPEEYPEIRDAADHIYLSQDLGMRKPEARIYQHVLQAEGFSPSDIVFFDDNADNIEGANQLGITSILVKDKITIPDYFAK VLC (SEQ ID NO: 56)PH31 Escherichia coli MRILLSNDDGVHAPGIQTLAKALREFADVQVVAPDRNRSGASNSLTLESYjjG SLRIFTFENGDIAVQMGIPTDCVYLGVNALMRPRPDIVVSGINAGPNLGDDVIYSGTVAAAMEGRHLGFPALAVSLDGHKHYDTAAAVICSILRALCKEPLRTGRILNINVPDLPLDQIKGIRVIRCGTRHPADQVIPQQDPRGNILYWIGPPGGKCDAGPGTDFAAVDEGYVSITPLHVDLTAHSAQDVVSDWLNSVGVGTQW (SEQ ID NO: 57) PH32 Escherichia coliMYERYAGLIFDMDGTILDTEPTHRKAWREVLGHYGLQYDIQAMIALNGS YqaBPTWRIAQAIIELNQADLDPHALAREKTEAVRSMLLDSVEPLPLVDVVKSWHGRRPMAVGTGSESAIAEALLAHLGLRHYFDAVVAADHVKHHKPAPDTFLLCAQRMGVQPTQCVVFEDADFGIQAARAAGMDAVDVRLL (SEQ ID NO: 58) PH33Escherichia coli MRFYRPLGRISALTFDLDDTLYDNRPVILRTEREALTFVQNYHPALRSF YigBQNEDLQRLRQAVREAEPEIYHDVTRWRFRSIEQAMLDAGLSAEEASAGAHAAMINFAKWRSRIDVPQQTHDTLKQLAKKWPLVAITNGNAQPELFGLGDYFEFVLRAGPHGRSKPFSDMYFLAAEKLNVPIGEILHVGDDLTTDVGGAIRSGMQACWIRPENGDLMQTWDSRLLPHLEISRLASLTSLI (SEQ ID NO: 59) PH34Escherichia coli MHINIAWQDVDTVLLDMDGILLDLAFDNYFWQKLVPETWGAKNGVIPQE YrfGAMEYMRQQYHDVQHTLNWYCLDYWSEQLGLDICAMTTEMGPRAVLREDTIPFLEALKASGKQRILLTNAHPHNLAVKLEHTGLDAHLDLLLSTHTFGYPKEDQRLWHAVAEATGLKAERTLFIDDSEAILDAAAQFGIRYCLGVINPDSGIAEKQYQRHPSLNDYRRLIPSLM (SEQ ID NO: 60) PH35 Escherichia coliMSTPRQILAAIFDMDGLLIDSEPLWDRAELDVMASLGVDISRRNELPDT GphLGLRIDMVVDLWYARQPWNGPSRQEVVERVIARAISLVEETRPLLPGVREAVALCKEQGLLVGLASASPLHMLEKVLTMFDLRDSFDALASAEKLPYSKPHPQVYLDCAAKLGVDPLTCVALEDSVNGMIASKAARMRSIVVPAPEAQNDPRFVLADVKLSSLTELTAKDLLG (SEQ ID NO: 61) PH36 Escherichia coliMSVKVIVTDMDGTFLNDAKTYNQPRFMAQYQELKKRGIKFVVASGNQYY YbiVQLISFFPELKDEISFVAENGALVYEHGKQLFHGELTRHESRIVIGELLKDKQLNFVACGLQSAYVSENAPEAFVALMAKHYHRLKPVKDYQEIDDVLFKFSLNLPDEQIPLVIDKLHVALDGIMKPVTSGFGFIDLIIPGLHKANGISRLLKRWDLSPQNVVAIGDSGNDAEMLKMARYSFAMGNAAENIKQIARYATDDNNHEGALNVIQAVLDNTSPFNS (SEQ ID NO: 62) PH37 Escherichia coliMAIKLIAIDMDGILLLPDHTISPAVKNAIAAARARGVNVVLITGRPYAG YidAVHNYLKELHMEQPGDYCITYNGALVQKAADGSTVAQTALSYDDYRFLEKLSREVGSHFHALDRITLYTANRDISYYTVHESFVATIPLVFCEAEKMDPNTQFLKVMMIDEPAILDQAIARIPQEVKEKYTVLKSAPYFLEILDKRVNKGTGVKSLADVLGIKPEEIMAIGDQENDIAMIEYAGVGVAMDNAIPSVKEVANFVTKSNLEDGVAFAIEKYVLN (SEQ ID NO: 63) PH38 Escherichia coliMITRVIALDLDGILLTPKKILLPSSIEALARAREAGYRLIIVTGRHHVA YbhAIHPFYQALALDTPAICCNGTYLYDYHAKTVLEADPMPVNKALQLIEMLNEHHIHGLMYVDDAMVYEHPIGHVIRTSNWAQTLPPEQRPTFTQVASLAETAQQVNAVWKFALTHDDLPQLQHFGKHVEHELGLECEWSWHDQVDIARGGNSKGKRLIKWVEAQGWSMENVVAFGDNENDISMLEAAGIGVAMGNADDAVKARANIVIGDNITDSIAQFIYSHLI (SEQ ID NO: 64) PH39 Escherichia coliMRFYRPLGRISALTFDLDDTLYDNRPVILRTEREALTFVQNYHPALRSF YbjIQNEDLQRLRQAVREAEPEIYHDVTRWRFRSIEQAMLDAGLSAEEASAGAHAAMINFAKWRSRIDVPQQTHDTLKQLAKKWPLVAITNGNAQPELFGLGDYFEFVLRAGPHGRSKPFSDMYFLAAEKLNVPIGEILHVGDDLTTDVGGAIRSGMQACWIRPENGDLMQTWDSRLLPHLEISRLASLTSLI (SEQ ID NO: 65) PH40Escherichia coli MYQVVASDLDGILLSPDHILSPYAKETLKLLTARGINFVFATGRHHVDV YigLGQIRDNLEIKSYMITSNGARVHDLDGNLIFAHNLDRDIASDLFGVVNDNPDIITNVYRDDEWFMNRHRPEEMRFFKEAVFQYALYEPGLLEPEGVSKVFFICDSHEQLLPLEQAINARWGDRVNVSFSTLICLEVMAGGVSKGHALEAVAKKLGYSLKDCIAFGDGMNDAEMLSMAGKGCIMGSAHQRLKDLHPELEVIGTNADDAVPHYLRKLYLS (SEQ ID NO: 66) PH41 Escherichia coliMTEPLIETPELSAKYAWFFDLDGILAEIKPHPDQVVVPDNILQGLQLLA OtsBTASDGALALISGRSMVELDALAKPYRFPLAGVHGAERRDINGKTHIVHLPDAIARDISVQLHTVIAQYPGAELEAKGMAFALHYRQAPQHEDALMTLAQRITQIWPQMALQQGKCVVEIKPRGISKGEAIAAFMQEAPFIGRIPVFLGDDLTDESGFAVVNRLGGMSVKIGTGATQASWRLAGVPDVWSWLEMITTALQQKRENNRSDDYESFSRSI (SEQ ID NO: 67) PH42 Escherichia coliMAKSVPAIFLDRDGTINVDHGYVHEIDNFEFIDGVIDAMRELKKMGFAL YaeDVVVINQSGIARGKFTEAQFETLIEWMDWSLADRDVDLDGIYYCPHHPQGSVEEFRQVCDCRKPHPGMLLSARDYLHIDMAASYMVGDKLEDMQAAVAANVGIKVLVRIGKPITPEAENAADWVLNSLADLPQAIKKQQKPAQ (SEQ ID NO: 68)

Accordingly, in one embodiment, provided herein is a recombinantmicroorganism that comprises an endogenous and/or exogenous nucleic acidmolecules capable of converting a carbon source to glyceraldehyde3-phosphate (G3P); at least one endogenous or exogenous nucleic acidmolecule encoding a (5-formylfuran-3-yl)methyl phosphate synthase thatcatalyzes the conversion of G3P to (5-formylfuran-3-yl)methyl phosphate;at least one endogenous or exogenous nucleic acid molecule encoding aphosphatase that catalyzes the conversion of (5-formylfuran-3-yl)methylphosphate to 4-HMF.

2,4-FDCA

In one embodiment, the present disclosure provides a recombinantmicroorganism capable of producing 2,4-furandicarboxylic acid (2,4-FDCA)from a carbon source. Some embodiments of the present disclosure arepresented in FIG. 1 , FIG. 2 , and FIG. 3 , which collectively detailthe biosynthetic conversion of a carbon feedstock to 2,4-FDCA.

In one embodiment, the recombinant microorganism comprises the novelcapacity to convert G3P to 2,4-FDCA via several enzymatically-catalyzedsuccessive steps described herein. In one embodiment, the presentdisclosure comprises converting 4-HMF to 2,4 FDCA directly or throughthe production of intermediates furan-2,4-dicarbaldehyde,4-(hydroxymethyl)furoic acid, 4-formylfuran-2-carboxylate,4-formylfuran-2-carboxylate, 2-formylfuran-4-carboxylate.

In one embodiment, the present disclosure comprises converting 4-HMF tofuran-2,4-dicarbaldehyde (Step D) and/or 4-(hydroxymethyl)furoic acid(Step E); converting furan-2,4-dicarbaldehyde to4-formylfuran-2-carboxylate (Step G) and/or 2-formylfuran-4-carboxylate(Step F) and/or converting 4-(hydroxymethyl)furoic acid to4-formylfuran-2-carboxylate (Step H); converting4-formylfuran-2-carboxylate to 2,4-FDCA (Step J) and/or converting2-formylfuran-4-carboxylate to 2,4-FDCA (Step I).

In one embodiment, the dehydrogenase is classified as EC number 1.1.1.when oxidizing an alcohol to a carbonyl group or EC number 1.2.1. whenoxidizing an carbonyl to acid. In some aspects, the dehydrogenase is analcohol dehydrogenase or an aldehyde dehydrogenase.

In some aspects, the oxidase from (c) is classified as EC number 1.1.3.In some aspects, the oxidase is 5-hydroxymethylfurfural oxidase. In someaspects the 5-hydroxymethylfurfural oxidase convert the4-hydroxymethylfurfural (4-HMF) into 2,4 FDCA in a three-step reaction.

In a further embodiment, the one or more carbon sources may includeglycerol or a monosaccharide.

In one embodiment, a microorganism comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, an oxidase, ora peroxygenase that catalyzes the conversion of 4-HMF tofuran-2,4-dicarbaldehyde and/or 4-(hydroxymethyl)furoic acid; at leastone endogenous or exogenous nucleic acid molecule encoding adehydrogenase, an oxidase, or a peroxygenase that catalyzes theconversion of furan-2,4-dicarbaldehyde to 4-formylfuran-2-carboxylateand/or 2-formylfuran-4-carboxylate and/or the conversion of4-(hydroxymethyl)furoic acid to 4-formylfuran-2-carboxylate; at leastone endogenous or exogenous nucleic acid molecule encoding adehydrogenase, an oxidase, or a peroxygenase that catalyzes theconversion of 2-formylfuran-4-carboxylate to 2,4-FDCA and/or4-formylfuran-2-carboxylate to 2,4-FDCA.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, an oxidase, ora peroxygenase that catalyzes the conversion of 4-HMF tofuran-2,4-dicarbaldehyde. In one embodiment, the dehydrogenase isclassified as EC number 1.1.1. In one embodiment, the dehydrogenase ECnumber 1.1.1 selected from alcohol dehydrogenase (EC number 1.1.1.1), oralcohol dehydrogenase (NADP+) (EC number 1.1.1.2), or D-xylose reductase(EC number 1.1.1.307), or aryl-alcohol dehydrogenase (EC number1.1.1.90), or aryl-alcohol dehydrogenase (NADP+) (EC number 1.1.1.91).In one embodiment the dehydrogenases can be derived from enzymecandidates listed at Table 3. In some embodiments, the dehydrogenasesare homologous or similar to the enzymes listed at Table 3. In someembodiments the 4-HMF dehydrogenase enzyme is encoded by an amino acidsequence listed in Table 3. In some embodiments, a dehydrogenase isevolved or engineered to improve its catalytic efficiency against itsdesirable substrate.

TABLE 3 4 HMF Dehydrogenases enzymes Name Organism Sequence DH1Zymomonas mobilis MLNFDYYNPTHIVFGKGRIAQLDILLSKDARVLVLYGGSSAQKTGILDEVRKALGDRTYFEFGGIEPNPSYETLMKAVEQVKQEKVDFLLAVGGGSVIDGIKEVAAAVPYEGEPWEILETDGKKIKEALPVGIVLILPATGSEMNRNSVVIRKSIKSKRGFHNDHVFPVFSILDPIKVYTLPPRQLANGVVDSFIHITEQYLTYPVDGMVQDEFAEGLLRTLIKIGPELLKDQKNYDLAANFMWTATLALNGLIGAGVPQDWATHMVGHELTAAFGIDHGRTLAIILPSLLQNQREAKKGKLLQYAKNVWHIDQGSDDERIDAAIEKTRHFFESLGIPTHLKDYDVGEESIDMLVKELEAHGMSQLGEHKAITPEVSRAILLASL (SEQ ID NO: 69) DH2Zymomonas mobilis MLNFDYYNPTHIAFGKDSIAKLDTLIPQDACVMVLYGGSSAKKTGILDEsubsp. pomaceae VKTALGSRKIHEFGGIEPNPSYETLMQAVEQVKKEKIDFLLAVGGGSVIATCC 29192 DGIKEVAAAVPYEGEPWEILETDGKKIKKALPLGTVLILPATGSEMNPNSVVIRKSIKAKRAFHNKIVFPLFSILDPIKVYTLPPRQIANGIVDSFVHITEQYLTYPVEGMVQDEFAEGLLRILINIGPKLLKDQKNYDLAANFMWTATLALNGLIGAGVPQDWATHMIGHEITAAFGVDHGRTLAIILPSLLQNQRQVKKDKLLQYAKNVWHIESGSEKERIDAVIAKTRSFFEEMGIPTHLSDYNIGKESIDMLIHELEAHGMTKLGEHNAITPDVSRAILIASL (SEQ ID NO: 70) DH3Shewanella baltica MLNFNYYNPTRIRFGKDTIAEIDTLVPSDAKVMILFGGSSARKTGILDEVKQSLGNRFIVEFDGIEPNPTYETLMKAVAQVREQKIDFLLAVGGGSVIDGIKEVAAAAVFEGEPWDILTSWGAKVTQAMPFGSVLILPAIGSEMNNASVVIRKSLQAKLPFRNDLVYPQFSILDPIKTFTLPERQVANGVVDAFVHITEQYLTYPVNAAVQDRFAEGLLQTLIELGPQVLAQPEDYDIRANLMWVATMALNGTIGVGVPHDWATHMIGHELTALYDIDHARTLAIVLPALLQCTKEAKREKLLQYADRVWHINTGIDDERIDAAIAKTKAFFEAMGIPTHLSAYDLDASHVDTLVKQLELHGMVALGEHGNINPAMSRDILTLAL (SEQ ID NO: 71) DH4Burkholderia MLNFDFYNPTRIVFGEKTAARLNDLLPAAARVLVLYGGESARSNGTLDEpseudoomallei VRAALGARDVREFGGIEPNPAYETLMRAVELARRERVDFLLAVGGGSVIDGIKEVAAAVPFEGDPWTILETHGANVAAALPFGCVLILPATGSEMNNGAVLIRRATRAKLAFRHPLVEPTESILDPIKTYTLPPRQVANGVVDAFTHIVEQYLTYPADGLAQDRFAEGLLQTLIEIGPKALAEPRDYATRANLMWVATLALNGLIGAGVPQDRATHMVGHELTARYDIDHARTLAVVLPSMLDVRRDAKRAKLLQYAARVWNIVDGPEDARIDAAIARTRAFFESLGVKIRLADYGVGADAIDGLIAQLEAHGMTRLGERKDVILDVSRRVLEASL (SEQ ID NO: 72) DH5Saccharomyces MSIPETQKGVIFYESHGKLEYKDIPVPKPKANELLINVKYSGVCHTDLHcerevisiae AWHGDWPLPTKLPLVGGHEGAGVVVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNEPNCPHADSSGYTHDGSFQQYATADAVQAAHIPQGTDLAEVAPVLCAGITVYKALKSANLMAGHWVAISGAAGGLGSLAVQYAKAMGYRVLGIDGGEGKEELFRSIGGEVFIDFIKEKDIVGAVLKAIDGGAHGVINVSVSEAAIEASTRYVRANGTTVLVGMPAGAKCCSDVFNQVVKSISIVGSCVGNRADTREALDFFARGLVKSPIKVVGLSILPEIYEKMEKGQIVGRYV VDTSK (SEQ ID NO: 73)DH6 Saccharomyces MSYPEKFEGIAIQSHEDWKNPKKTKYDPKPFYDHDIDIKIEACGVCGSDcerevisiae IHCAAGHWGNMKMPLVVGHEIVGKVVKLGPKSNSGLKVGQRVGVGAQVFSCLECDRCKNDNEPYCTKFVTTYSQPYEDGYVSQGGYANYVRVHEHFVVPIPENIPSHLAAPLLCGGLTVYSPLVRNGCGPGKKVGIVGLGGIGSMGTLISKAMGAETYVISRSSRKREDAMKMGADHYIATLEEGDWGEKYFDTFDLIVVCASSLTDIDFNIMPKAMKVGGRIVSISIPEQHEMLSLKPYGLKAVSISYSALGSIKELNQLLKLVSEKDIKIWVETLPVGEAGVHEAFERMEKGDVRYRFTLVGYDKEFSD (SEQ ID NO: 74) DH7 Pseudomonas putidaMSIEHRLNHIAGQLSGNGEVLLNSVDAHTGEPLPYAFHQATSDEVDAAVQAAEAAYPAYRSTSPAQRAAFLDAIANELDALGDDFVQHVMRETALPEARIRGERARTSNQLRLFADVVRRGDFLGARIDRAQPERTPLPRPDLRQYRIGVGPVAVFGASNFPLAFSTAGGDTASALAAGCPVVFKAHSGHMLTAAHVAAAIDRAVAGSGMPAGVFNMIYGAGVGEVLVKHPAIQAVGFTGSLRGGRALCDMAAARPQPIPVFAEMSSINPVIVLPQALQARGEQVAGELAASVVLGCGQFCTNPGLVVGIKSPQFERFVHTLVARMADQAPQTMLNAGTLRSYQSGVQHLLAHPGIQHLAGQPQAGKQAQPQLFKADVSLLLDSDPLLQEEVFGPTTVVVEVADAQQLAEALRHLQGQLTATLIAEPDDLRAFAALVPLLERKAGRLLLNGYPTGVEVSDAMVHGGPYPATSDARGTSVGTLAIDRFLRPVCFQNYPDALLPEALKSANPLGIARLVDGVASRGAV (SEQ ID NO: 75) DH8Pseudomonas putida MSIEHRLNHIAGQLSGNGDVLLNSVDAHTGEPLPYAFHQATGDEVEAAVQAADAAYPAYRSTSPAQRAAFLDAIANELDALGDDFIQHVMRETALPEARIRGERSRTSNQLRLFAEVVRRGDFYAARIDRALPQRTPLPRPDLRQYRIGVGPVAVFGASNFPLAFSTAGGDTASALAAGCPVVFKAHSGHMLTAAHVAGAIDRAVATSGMPAGVFNLIYGAGVGEALVKHPAIQAVGFTGSLRGGRALCDMAAARPQPIPVFAEMSSINPVIVLPQALQARGEQVAGELAASVVMGCGQFCTNPGLVVGIQSPQFEHFVQTLVARMADQGPQTMLNAGTLRSYQNGVQHLLAHPGIQHLAGQPHTGNQAQPQLFKADVSLLLNGDPLLQEEVFGPTTVVVEVADAEQLAEALRHLQGQLTATLIAEPDDLRAFASLVPLLERKAGRLLLNGYPTGVEVSDAMVHGGPYPATSDARGTSVGTLAIDRFLRPVCFQNYPDALLPDALKNANPLGIARLLDGVNSRDAV (SEQ ID NO: 76) DH9 Pseudomonas sp.MSIEHRLNHIAGQLSGHGDVLLHSLDAHTGEALPYAFHQATGDEVEAAA NBRC 111139QAAEVAYPSYRSTRPDQRAAFLDAIASELDALGDDFIQDVMRETALPEARIRGERSRTSNQLRLFAEVVRRGDFYAARIDRALPQRTPLPRPDLRQYRIGVGPVAVFGASNFPLAFSTAGGDTASALAAGCPVVFKAHSGHMLTAAHVAAAIDRAVTGSGMPAGVFNMIYGAGVGEALVKHPAIQAVGFTGSLRGGRALCDMAAARPQPIPVFAEMSSINPVIVLPQALQARGEQVATELAASVVLGCGQFCTNPGLVVGIRSPHFEHFLQTLVARMADQGPQTMLNAGTLRSYQNAVQHLLAHPGIQHLAGQPQTGNQAQPQLFKADVSLLLNGDPLLQEEVFGPCTVVVEVADAQQLAEALRHLQGQLTATLIAEPDDLRAFASLVPLLERKAGRLLLNGYPTGVEVSDAMVHGGPYPATSDARGTSVGTLAIDRFLRPVCFQNYPDALLPDALKNANPLGIARLLEGVSSREAV (SEQ ID NO: 77) DH10Pseudomonas sp. MQIQGKNYIGGARSGEGEVRVYSIDATTGEKLPYEFFQASTAEVDAAAR JUb52AAEQAAPLYRKLSAEQRATFLDAIADELDALGDDFVQLVCQETALPAGRIQGERGRTSGQMRLFAKVLRRGDFHGARIDTALPERKPLPRPDLRQYRIGLGPVAVFGASNFPLAFSTAGGDTAAALAAGCPVVFKAHSGHMVTAEYVADAIIRAAEKTGMPKGVFNMIYGGGVGEQLVKHPAIQAVGFTGSLRGGRALCDMAAARPQPIPVFAEMSSINPVVVLPEALKARGDAITGELAASVVLGCGQFCTNPGLVIGLRSPEFSTFLEGLAAAMNEQAPQTMLNPGTLKSYEKGVAALLAHSGVQHLAGANQEGNQARPQLFKADVSLLLENDELLQEEVFGPTTVVVEVADEAQLHQALQGLHGQLTATLLAEPADLQRFEAIIGLLEQKAGRLLLNGYPTGVEVCDAMVHGGPYPATSDARGTSVGTLAIDRFLRPVCYQNYPDAFLPEALQNANPLGIQRLVNGENTKAAI (SEQ ID NO: 78) DH11 PseudomonasMFGHNFIGGARTAQGNLTLQSLDAGTGEALPYSFHQATPEEVDAAALAA citronellolisEAAFPAYRALPDARRAEFLDAIAAELDALGEDFIAIVCRETALPAARIQGERARTSNQLRLFAQVLRRGDYHGARIDRALPERQPLPRPDLRQCRIGVGPVAVFGASNFPLAFSTAGGDTAAALAAGCPVVFKAHSGHMATAEHVASAIVRAAQATGMPAGVFNMIYGGGVGERLVKHPAIQAVGFTGSLKGGRALCDLAAARPQPIPVFAEMSSINPVLALPAALAARGEQVAADLAASVVLGCGQFCTNPGMVIGIASAEFSAFVASLTGRMADQPAQTMLNAGTLKSYERGIAALHAHPGIRHLAGQPQKGRQALPQLFQADARLLIEGDELLQEEVFGPVTVVVEVADAAELQRALQGLRGQLTATLIAEPEDLSCFAALVPLLERKAGRLLLNGYPTGVEVCDAMVHGGPYPATSDARGTSVGTLAIDRFLRPVCYQNYPDALLPPALKDANPLGIARLVDGVASREPL (SEQ ID NO: 79)

In one embodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the HMF oxidase can bederived from the gene hmfH. In some embodiments, HMF oxidase can bederived from Methylovorus sp. MP688 or Cupriavidus basilensis. SeeDijkman and Fraaije (2014. Applied Environmental Microbiology,80.3:1082-1090) and Koopman et al. (2010. PNAS, 107(11):4919-4924). Inone embodiment, the HMF oxidase EC number 1.1.3 is aryl-alcohol oxidase(EC number 1.1.3.7). See Carro et al. (2015). In one embodiment, theperoxygenase is classified as EC number 1.11.2. In one embodiment, theperoxygenase EC number 1.11.2 is unspecific peroxygenase (EC number1.11.2.1). See Carro et al. (2015). In some embodiments, the HMF oxidasecan be derived from enzyme candidates listed at Table 4. In someembodiments, the HMF oxidase is homologous or similar to the enzymeslisted at Table 4. In some embodiments the 4-HMF oxidaze enzyme isencoded by an amino acid sequence listed in Table 4. In someembodiments, the HMF oxidase enzyme is evolved or engineered to improveits catalytic efficiency (See Martin et al. Biotechnology for Biofuels.(2018) 11, Article number: 56).

TABLE 4 4-HMF oxidases enzymes Name Organism Sequence HmfH1Methylovorus sp MTDTIFDYVIVGGGTAGSVLANRLSARPENRVLLIEAGIDTPENNIPPEIHDGLRPWLPRLSGDKFFWPNLTIHRAAEHPGITREPQFYEQGRLLGGGSSVNMVVSNRGLPRDYDEWQALGADGWDWQGVLPYFIKTERDADYGDDPLHGNAGPIPIGRVDSRHWSDFTVAATQALEAAGLPNIHDQNARFDDGYFPPAFTLKGEERFSAARGYLDASVRVRPNLSLWTESRVLKLLTTGNAITGVSVLRGRETLQVQAREVILTAGALQSPAILLRTGIGPAADLHALGIPVLADRPGVGRNLWEHSSIGVVAPLTEQARADASTGKAGSRHQLGIRASSGVDPATPSDLFLHIGADPVSGLASAVFWVNKPSSTGWLKLKDADPFSYPDVDFNLLSDPRDLGRLKAGLRLITHYFAAPSLAKYGLALALSRFAAPQPGGPLLNDLLQDEAALERYLRTNVGGVWHASGTARIGRADDSQAVVDKAGRVYGVTGLRVADASIMPTVPTANTNLPTLMLAEKIADAILTQA (SEQ ID NO: 80) HmfH2Cupriavidus MDTPRERFDYVIVGGGSAGCVLANRLSQDPAIRVALIEAGVDTPPDAVP basilensisAEILDSYPMPLFFGDRYIWPSLQARAVAGGRSKVYEQGRVMGGGSSINVQAANRGLPRDYDEWAASGASGWSWQDVLPYFRHLERDVDYGNSPLHGSHGPVPIRRILPQAWPPFCTEFAHAMGRSGLSALADQNAEFGDGWFPAAFSNLDDKRVSTAIAYLDADTRRRANLRIYAETTVRKLVVSGREARGVIAMRADGSRLALDAGEVIVSAGALQSPAILMRAGIGDAGALQALGIEVVADRPGVGRNLQDHPALTFCQFLAPQYRMPLSRRRASMTAARFSSGVPGGEASDMYLSSSTRAGWHALGNRLGLFFLWCNRPFSRGQVSLAGAQPDVPPMVELNLLDDERDLRRMVAGVRKLVQIVGASALHQHPGDFFPATFSPRVKALSRVSRGNVLLTELLGAVLDVSGPLRRSLIARFVTGGANLASLLTDESALEGFVRQSVFGVWHASGTCRMGAHADRSAVTDAAGRVHDVGRLRVIDASLMPRLPTANTNIPTIMLAEKIADTMQAERRAVRPASSEVAHPS (SEQ ID NO: 81) HmfH3Cupriavidus MDTPRERFDYVIVGGGSAGCVLANRLSQDPAIRVALIEGGVDTPPDAVP necatorVEILDSYPMPLFFGDRYIWPSLQARAVAGGRSKVYEQGRVMGGGSSINVQAANRGLPRDYDEWAASGAPGWSWQDVLPYFRNLERDVDYGNSPLHGSHGPVPIRRILPQAWPPFCTEFAHAMGLSGLSALADQNAEFGDGWFPAAFSNLDDKRVSTAIAYLDADIRRRANLRIYAETTVRKLVVSGREARGVIAIRADGSRLALDAGEVIVSAGALQSPAILMRAGIGDAGALQALGIEVVADRPGVGRNLQDHPALTFCQFLAPQYRMPLSRRRASMTAARFSSGVPGGEASDMYLSSSTRAGWHALGNRLGLFFLWCNRPFSRGQVSLAGAQPDVPPMVELNLLDDERDLRRMVAGVRKLVQIVGASALHQHPGDFFPATFSPRVKALSRLSRGNALLTELLGALLDVSGPLRRSLIARFVTGGANLASLLVEESALEGFVRQSVFGVWHASGTCRMGAHADRSAVTDAAGRVHDVGRLRVVDASLMPRLPTANTNIPTIMLAEKIADTMQAERRAVRLASSEVAHQS (SEQ ID NO: 82) HmfH4Cupriavidus MGTPRDRFDYVIVGGGSAGCVLANRLSRDPGIRVALIEGGVDTPPGAVPpinatubonensis AEILDSYPMPLFFGDRYLWPSLQARAVAGGRARLYEQGRVMGGGSSINVQAANRGLPRDYDEWAASGAPGWSWQEVLPYFRKLERDVDFASSPMHGSDGPVPIRRILPPAWPPFCTAFAQAMGRSGLSALDDQNAEFGDGWFPAAFSNLDGKRVSTAIAYLDANTRKRTNLRIFAETTVKELVVSGREARGVIAVRADGARLALEAAEVIVSAGALQSPAILMRAGIGDAAALQALGIEVVADRPGVGRNLQDHPALTFCQFLAPEYRMPLARRRSSMTAARFSSEVPGGEASDMYLSSSTRAGWHALGNRLGLFFLWCNRPFSRGQVSLAGAQPEVSPLVELNLLDDERDLRRMVAGVRRLVRIVGASALHQHPDDFFPAIFSPRVKAMSRVSPGNALLTALLGALLDVSGPLRRSLIARFVTGGANLASLLADESALEGFVRQSVFGVWHASGTCRMGAHADRSAVTDTTGRVHDVGRLRVVDASLMPRLPTANTNIPTIMLAEKIADAMLAERRATRRALSEVADPG (SEQ ID NO: 83) HmfH5Pandoraea sp. B-6 MPRGHAHRRIRRHSVQNVRERFDYVIIGGGSAGCVLAHRLSANRELRVALIEAGSDTPPGAIPAEILDSYPMPVFCGDRYIWPELKAKATAASPLKVYEQGKVMGGGSSINVQAANRGLPRDYDDWAEQGASGWAWKDVLPYFRKLERDADYGGSALHGADGPVAIRRIKPDAWPRFCHAFAEGLQRNGLPMLEDQNAEFGDGMFPAAFSNLDDKRVSTAVAYLDAATRARTNLRIYSNTIVERLIVTGQRAHGVVAMSAGGERLQIDAAEVIVSAGALQSPALLLRAGIGAGSELQALGIPVVADRPGVGRNLQDHPSLTFCHFLDPEFRMPLSRRRASMTAARFSSGLDGCDNADMYLSSATRAAWHALGNRLGLFFLWCNRPFSRGRVQLTSADPFTPPRVDLNLLDDERDARRMAIGVRRVAQIVQQTALHRHPDDFFPAAFSPRVKALSRFSAGNAALTKVLGLALDTPAPLRRWIIDTFVTGGIRMSALLADDKELDAFIRKYVEGVWHASGICRMGPASDRMAVINQEGLVHDVANLRVVDASLMPKLPSANTNIPTIMMAEKIADAILARRKAPPGVLVS SEA (SEQ ID NO: 84)HmfH6 Methylovorus sp MIDTIFDYVIVGGGTAGSVLANRLSARPENRVLLIEAGIDTPENNIPPEIHDGLRPWLPRLSGDKFFWPNLTIHRAAEHPGITREPQFYEQGRLLGGGSSVNMVVSNRGLPRDYDEWQALGADGWDWQGVLPYFIKTERDADYGDDPLHGNAGPIPIGRVDSRHWSDFTVAATQALEAAGLPNIHDQNARFDDGYFPPAFTLKGEERFSAARGYLDASVRVRPNLSLWIESRVLKLLTIGNAITGVSVLRGRETLQVQAREVILTAGALQSPAILLRTGIGPAADLHALGIPVLADRPGVGRNLWEHSSIGVVAPLTEQARADASIGKAGSRHQLGIRASSGVDPATPSDLFLHIGADPVSGLASARFWVNKPSSTGWLKLKDADPFSYPDVDFNLLSDPRDLGRLKAGLRLITHYFAAPSLAKYGLALALSRFAAPQPGGPLLNDLLQDEAALERYLRINVGGVFHASGTARIGRADDSQAVVDKAGRVYGVTGLRVADASIMPTVPTANTNLPTLMLAEKIADAILTQA (SEQ ID NO: 85) HmfH7Methylovorus sp MIDTIFDYVIVGGGTAGSVLANRLSARPENRVLLIEAGIDTPENNIPPE MUTIHDGLRPWLPRLSGDKFFWPNLTVYRAAEHPGITREPQFYEQGRLLGGGSSVNMVVSNRGLPRDYDEWQALGADGWDWQGVLPYFIKTERDADYGDDPLHGNAGPIP1GRVDSRHWSDFTVAATQALEAAGLPNIHDQNARFDDGYFPPAFTLKGEERFSAARGYLDASVRVRPNLSLWTESRVLKLLTTGNATTGVSVLRGRETLQVQAREVILTAGALQSPAILLRTGIGPAADLHALGIPVLADRPGVGRNLWEHSSIGVVAPLTEQARADASTGKAGSRHQLGIRASSGVDPATPSDLFLHIHADPVSGLASARFWVNKPSSTGWLKLKDADPFSYPDVDFNLLSDPRDLGRLKAGLRLIKHYFAYPSLAKYGLALALSRFEAPQPGGPLLNDLLQDEAALERYLRTNVGGVFHASGTARIGRADDSQAVVDKAGRVYGVTGLRVADASIMPTVPTANTNLPTLMLAEKIADAILTQA (SEQ ID NO: 86)

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion of 4-HMF to4-(hydroxymethyl)furoic acid. In one embodiment, the dehydrogenase isclassified as EC number 1.2.1. In one embodiment, the dehydrogenase ECnumber 1.2.1 selected from aldehyde dehydrogenase (NAD+) (EC number1.2.1.3) or aldehyde dehydrogenase (NADP+) (EC number 1.2.1.4) oraldehyde dehydrogenase [NAD(P)+] (EC number 1.2.1.5) or4-(γ-glutamylamino)butanal dehydrogenase (EC number 1.2.1.99). In oneembodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. In oneembodiment, the oxidase EC number 1.1.3 is aryl-alcohol oxidase (ECnumber 1.1.3.7). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1).

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion offuran-2,4-dicarbaldehyde to 4-formylfuran-2-carboxylate and/or to2-formylfuran-4-carboxylate. In one embodiment, the dehydrogenase isclassified as EC number 1.2.1. In one embodiment, the dehydrogenase ECnumber 1.2.1 selected from aldehyde dehydrogenase (NAD+) (EC number1.2.1.3) or aldehyde dehydrogenase (NADP+) (EC number 1.2.1.4) oraldehyde dehydrogenase [NAD(P)+] (EC number 1.2.1.5) or4-(γ-glutamylamino)butanal dehydrogenase (EC number 1.2.1.99). In oneembodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. In oneembodiment, the oxidase EC number 1.1.3 is aryl-alcohol oxidase (ECnumber 1.1.3.7). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1).

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion of4-(hydroxymethyl)furoic acid to 4-formylfuran-2-carboxylate. In oneembodiment, the dehydrogenase is classified as EC number 1.1.1. In oneembodiment, the dehydrogenase EC number 1.1.1 selected from alcoholdehydrogenase (EC number 1.1.1.1), or alcohol dehydrogenase (NADP+) (ECnumber 1.1.1.2), or D-xylose reductase (EC number 1.1.1.307), oraryl-alcohol dehydrogenase (EC number 1.1.1.90), or aryl-alcoholdehydrogenase (NADP+) (EC number 1.1.1.91). In one embodiment, thedehydrogenase EC number 1.1.1 is. In one embodiment, the oxidase isclassified as EC number 1.1.3. In one embodiment, the oxidase EC number1.1.3 is 5-(hydroxymethylfurfural oxidase (EC number 1.1.3.47). In someembodiments the EC 1.1.3.47 oxidase can be derived from the gene hmfH.In some embodiments, hmfH can be derived from Methylovorus sp. MP688 orCupriavidus basilensis. In one embodiment, the oxidase EC number 1.1.3is aryl-alcohol oxidase (EC number 1.1.3.7). In one embodiment, theperoxygenase is classified as EC number 1.11.2. In one embodiment, theperoxygenase EC number 1.11.2 is unspecific peroxygenase (EC number1.11.2.1).

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion of4-formylfuran-2-carboxylate and/or 2-formylfuran-4-carboxylate to2,4-FDCA. In one embodiment, the dehydrogenase is classified as ECnumber 1.2.1. In one embodiment, the dehydrogenase EC number 1.2.1selected from aldehyde dehydrogenase (NAD+) (EC number 1.2.1.3) oraldehyde dehydrogenase (NADP+) (EC number 1.2.1.4) or aldehydedehydrogenase [NAD(P)+] (EC number 1.2.1.5) or4-(γ-glutamylamino)butanal dehydrogenase (EC number 1.2.1.99). In oneembodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. In oneembodiment, the oxidase EC number 1.1.3 is aryl-alcohol oxidase (ECnumber 1.1.3.7). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1).

In some aspects, 2,4-FDCA is produced enzymatically, in the absence ofmicrobes. In some aspects, 2,4-FDCA is produced enzymatically in one ormore vessels. In some aspects, the one or more vessels are substantiallyfree of microbes. In some aspects, the enzymatic production of 2,4-FDCAis performed in the same step-wise fashion as described with in themethods utilizing recombinant microorganisms, but substantially free ofmicroorganisms or in the absence of microorganisms. In some aspects, theenzymes utilized in the enzymatic production of 2,4-FDCA are isolatedfrom microbes, recombinant or otherwise, and provided to theircorresponding substrates for the stepwise production of theintermediates utilized to produce 2,4-FDCA. In some aspects, one or moreof the steps of the methods are performed in the same vessel. In someaspects, once the desired product is produced as a result of theindividual method steps described herein, the product is isolated andpurified and then utilized as the substrate in the next step of themethod of producing 2,4-FDCA.

2,4-furandimethanol

In one embodiment, the present disclosure provides a recombinantmicroorganism capable of producing 2,4-furandimethanol from a carbonsource. Some embodiments of the present disclosure are presented in FIG.1 , FIG. 2 , and FIG. 3 , which collectively detail the biosyntheticconversion of a carbon feedstock to 2,4-furandimethanol.

In one embodiment, the bioproduction of 2,4-furandimethanol from 4-HMFis catalyzed by a dehydrogenase encoded by the microorganism. In oneembodiment, the dehydrogenase is classified as EC number 1.1.1. In oneembodiment, the dehydrogenase EC number 1.1.1 is selected from alcoholdehydrogenase (EC number 1.1.1.1). In one embodiment, the dehydrogenaseEC number 1.1.1 is selected from alcohol dehydrogenase (NADP+) (ECnumber 1.1.1.2). In one embodiment, the dehydrogenase EC number 1.1.1 isselected from D-xylose reductase (EC number 1.1.1.90). In oneembodiment, the dehydrogenase EC number 1.1.1 is selected fromaryl-alcohol dehydrogenase (EC number 1.1.1.91). In one embodiment thedehydrogenases can be derived from enzyme candidates listed at Table 5.In some embodiments, the dehydrogenases are homologous or similar to theenzymes listed at Table 5. In some embodiments the 4-HMF reductaseenzyme is encoded by an amino acid sequence listed in Table 5. In someembodiments, a dehydrogenases is evolved or engineered to improve itscatalytic efficiency for 4-HMF reduction to 2,4-furandimethanol.

TABLE 5 4-HMF reductase enzymes (4-HMF reduction to 2,4-furandimethanol)Name Organism Sequence DH1 Zymomonas mobilisMLNFDYYNPTHIVFGKGRIAQLDILLSKDARVLVLYGGSSAQKTGILDEVRKALGDRTYFEFGGIEPNPSYETLMKAVEQVKQEKVDFLLAVGGGSVIDGIKEVAAAVPYEGEPWEILETDGKKIKEALPVGIVLILPATGSEMNRNSVVIRKSIKSKRGFHNDHVFPVFSILDPIKVYTLPPRQLANGVVDSFIHITEQYLTYPVDGMVQDEFAEGLLRTLIKIGPELLKDQKNYDLAANFMWTATLALNGLIGAGVPQDWATHMVGHELTAAFGIDHGRTLAIILPSLLQNQREAKKGKLLQYAKNVWHIDQGSDDERIDAAIEKTRHFFESLGIPTHLKDYDVGEESIDMLVKELEAHGMSQLGEHKAITPEVSRAILLASL (SEQ ID NO: 87) DH2Zymomonas mobilis MLNFDYYNPTHIAFGKDSIAKLDTLIPQDACVMVLYGGSSAKKTGILDEsubsp. pomaceae VKTALGSRKIHEFGGIEPNPSYETLMQAVEQVKKEKIDFLLAVGGGSVIATCC 29192 DGIKEVAAAVPYEGEPWEILETDGKKIKKALPLGTVLILPATGSEMNPNSVVIRKSIKAKRAFHNKIVFPLFSILDPIKVYTLPPRQIANGIVDSFVHITEQYLTYPVEGMVQDEFAEGLLRILINIGPKLLKDQKNYDLAANFMWTATLALNGLIGAGVPQDWATHMIGHEITAAFGVDHGRTLAIILPSLLQNQRQVKKDKLLQYAKNVWHIESGSEKERIDAVIAKTRSFFEEMGIPTHLSDYNIGKESIDMLIHELEAHGMTKLGEHNAITPDVSRAILIASL (SEQ ID NO: 88) DH3Shewanella baltica MLNFNYYNPTRIRFGKDTIAEIDTLVPSDAKVMILFGGSSARKTGILDEVKQSLGNRFIVEFDGIEPNPTYETLMKAVAQVREQKIDFLLAVGGGSVIDGIKEVAAAAVFEGEPWDILTSWGAKVTQAMPFGSVLILPAIGSEMNNASVVIRKSLQAKLPFRNDLVYPQFSILDPIKTFTLPERQVANGVVDAFVHITEQYLTYPVNAAVQDRFAEGLLQTLIELGPQVLAQPEDYDIRANLMWVATMALNGTIGVGVPHDWATHMIGHELTALYDIDHARTLAIVLPALLQCTKEAKREKLLQYADRVWHINTGIDDERIDAAIAKTKAFFEAMGIPTHLSAYDLDASHVDTLVKQLELHGMVALGEHGNINPAMSRDILTLAL (SEQ ID NO: 89) DH4Burkholderia MLNFDFYNPTRIVFGEKTAARLNDLLPAAARVLVLYGGESARSNGTLDEpseudoomallei VRAALGARDVREFGGIEPNPAYETLMRAVELARRERVDFLLAVGGGSVIDGIKEVAAAVPFEGDPWTILETHGANVAAALPFGCVLILPATGSEMNNGAVLIRRATRAKLAFRHPLVEPTESILDPIKTYTLPPRQVANGVVDAFTHIVEQYLTYPADGLAQDRFAEGLLQTLIEIGPKALAEPRDYATRANLMWVATLALNGLIGAGVPQDRATHMVGHELTARYDIDHARTLAVVLPSMLDVRRDAKRAKLLQYAARVWNIVDGPEDARIDAAIARTRAFFESLGVKTRLADYGVGADAIDGLIAQLEAHGMTRLGERKDVILDVSRRVLEASL (SEQ ID NO: 90) DH5Saccharomyces MSIPETQKGVIFYESHGKLEYKDIPVPKPKANELLINVKYSGVCHTDLHcerevisiae AWHGDWPLPTKLPLVGGHEGAGVVVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNEPNCPHADSSGYTHDGSFQQYATADAVQAAHIPQGTDLAEVAPVLCAGITVYKALKSANLMAGHWVAISGAAGGLGSLAVQYAKAMGYRVLGIDGGEGKEELFRSIGGEVFIDFIKEKDIVGAVLKAIDGGAHGVINVSVSEAAIEASTRYVRANGTTVLVGMPAGAKCCSDVFNQVVKSISIVGSCVGNRADTREALDFFARGLVKSPIKVVGLSTLPEIYEKMEKGQIVGRYV VDTSK (SEQ ID NO: 91)DH6 Saccharomyces MSYPEKFEGIAIQSHEDWKNPKKTKYDPKPFYDHDIDIKIEACGVCGSDcerevisiae IHCAAGHWGNMKMPLVVGHEIVGKVVKLGPKSNSGLKVGQRVGVGAQVFSCLECDRCKNDNEPYCTKFVTTYSQPYEDGYVSQGGYANYVRVHEHFVVPIPENIPSHLAAPLLCGGLTVYSPLVRNGCGPGKKVGIVGLGGIGSMGTLISKAMGAETYVISRSSRKREDAMKMGADHYIATLEEGDWGEKYFDTFDLIVVCASSLTDIDFNIMPKAMKVGGRIVSISIPEQHEMLSLKPYGLKAVSISYSALGSIKELNQLLKLVSEKDIKIWVETLPVGEAGVHEAFERMEKGDVRYRFTLVGYDKEFSD (SEQ ID NO: 92)

In some aspects, 2,4-furandimethanol is produced enzymatically, in theabsence of microbes. In some aspects, 2,4-furandimethanol is producedenzymatically in one or more vessels. In some aspects, the one or morevessels are substantially free of microbes. In some aspects, theenzymatic production of 2,4-furandimethanol is performed in the samestep-wise fashion as described with in the methods utilizing recombinantmicroorganisms, but substantially free of microorganisms or in theabsence of microorganisms. In some aspects, the enzymes utilized in theenzymatic production of 2,4-furandimethanol are isolated from microbes,recombinant or otherwise, and provided to their corresponding substratesfor the stepwise production of the intermediates utilized to produce2,4-furandimethanol. In some aspects, one or more of the steps of themethods are performed in the same vessel. In some aspects, once thedesired product is produced as a result of the individual method stepsdescribed herein, the product is isolated and purified and then utilizedas the substrate in the next step of the method of producing2,4-furandimethanol.

Furan-2,4-dicarbaldehyde

In one embodiment, the present disclosure provides a recombinantmicroorganism capable of producing furan-2,4-dicarbaldehyde from acarbon source. Some embodiments of the present disclosure are presentedin FIG. 1 , FIG. 2 , and FIG. 3 , which collectively detail thebiosynthetic conversion of a carbon feedstock tofuran-2,4-dicarbaldehyde.

In one embodiment, step D in FIG. 2 is a single step reaction utilizing4-HMF as a substrate. In one embodiment, the bioproduction offuran-2,4-dicarbaldehyde from 4-HMF is catalyzed by one or more enzymesrepresented by EC numbers 1.1.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, an oxidase, ora peroxygenase that catalyzes the conversion of 4-HMF tofuran-2,4-dicarbaldehyde. In one embodiment, the dehydrogenase isclassified as EC number 1.1.1. In one embodiment, the dehydrogenase ECnumber 1.1.1 selected from alcohol dehydrogenase (EC number 1.1.1.1), oralcohol dehydrogenase (NADP+) (EC number 1.1.1.2), or D-xylose reductase(EC number 1.1.1.307), or aryl-alcohol dehydrogenase (EC number1.1.1.90), or aryl-alcohol dehydrogenase (NADP+) (EC number 1.1.1.91).In one embodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. SeeDijkman and Fraaije (2014) and Koopman et al. (2010). In one embodiment,the oxidase EC number 1.1.3 is aryl-alcohol oxidase (EC number 1.1.3.7).See Carro et al. (2015). In one embodiment, the peroxygenase isclassified as EC number 1.11.2. In one embodiment, the peroxygenase ECnumber 1.11.2 is unspecific peroxygenase (EC number 1.11.2.1). See Carroet al. (2015).

In some aspects, furan-2,4-dicarbaldehyde is produced enzymatically, inthe absence of microbes. In some aspects, furan-2,4-dicarbaldehyde isproduced enzymatically in one or more vessels. In some aspects, the oneor more vessels are substantially free of microbes. In some aspects, theenzymatic production of furan-2,4-dicarbaldehyde is performed in thesame step-wise fashion as described with in the methods utilizingrecombinant microorganisms, but substantially free of microorganisms orin the absence of microorganisms. In some aspects, the enzymes utilizedin the enzymatic production of furan-2,4-dicarbaldehyde are isolatedfrom microbes, recombinant or otherwise, and provided to theircorresponding substrates for the stepwise production of theintermediates utilized to produce furan-2,4-dicarbaldehyde. In someaspects, one or more of the steps of the methods are performed in thesame vessel. In some aspects, once the desired product is produced as aresult of the individual method steps described herein, the product isisolated and purified and then utilized as the substrate in the nextstep of the method of producing furan-2,4-dicarbaldehyde.

4-(hydroxymethyl)furoic acid

In one embodiment, the present disclosure provides a recombinantmicroorganism capable of producing 4-(hydroxymethyl)furoic acid from acarbon source. Some embodiments of the present disclosure are presentedin FIG. 1 , FIG. 2 , and FIG. 3 , which collectively detail thebiosynthetic conversion of a carbon feedstock to 4-(hydroxymethyl)furoicacid.

In one embodiment, step E in FIG. 2 is a single step reaction utilizing4-HMF as a substrate. In one embodiment, the bioproduction of4-(hydroxymethyl)furoic acid from 4-HMF is catalyzed by one or moreenzymes represented by EC numbers 1.1.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion of 4-HMF to4-(hydroxymethyl)furoic acid. In one embodiment, the dehydrogenase isclassified as EC number 1.2.1. In one embodiment, the dehydrogenase ECnumber 1.2.1 selected from aldehyde dehydrogenase (NAD+) (EC number1.2.1.3) or aldehyde dehydrogenase (NADP+) (EC number 1.2.1.4) oraldehyde dehydrogenase [NAD(P)+] (EC number 1.2.1.5) or4-(γ-glutamylamino)butanal dehydrogenase (EC number 1.2.1.99). In oneembodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. In oneembodiment, the oxidase EC number 1.1.3 is aryl-alcohol oxidase (ECnumber 1.1.3.7). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1).

In some aspects, 4-(hydroxymethyl)furoic acid is produced enzymatically,in the absence of microbes. In some aspects, 4-(hydroxymethyl)furoicacid is produced enzymatically in one or more vessels. In some aspects,the one or more vessels are substantially free of microbes. In someaspects, the enzymatic production of 4-(hydroxymethyl)furoic acid isperformed in the same step-wise fashion as described with in the methodsutilizing recombinant microorganisms, but substantially free ofmicroorganisms or in the absence of microorganisms. In some aspects, theenzymes utilized in the enzymatic production of 4-(hydroxymethyl)furoicacid are isolated from microbes, recombinant or otherwise, and providedto their corresponding substrates for the stepwise production of theintermediates utilized to produce 4-(hydroxymethyl)furoic acid. In someaspects, one or more of the steps of the methods are performed in thesame vessel. In some aspects, once the desired product is produced as aresult of the individual method steps described herein, the product isisolated and purified and then utilized as the substrate in the nextstep of the method of producing 4-(hydroxymethyl)furoic acid.

2-formylfuran-4-carboxylate

In one embodiment, the present disclosure provides a recombinantmicroorganism capable of producing 2-formylfuran-4-carboxylate from acarbon source. Some embodiments of the present disclosure are presentedin FIG. 1 , FIG. 2 , and FIG. 3 , which collectively detail thebiosynthetic conversion of a carbon feedstock to2-formylfuran-4-carboxylate.

In one embodiment, step F in FIG. 2 is a single step reaction utilizingfuran-2,4-dicarbaldehyde as a substrate. In one embodiment, thebioproduction of 2-formylfuran-4-carboxylate fromfuran-2,4-dicarbaldehyde is catalyzed by one or more enzymes representedby EC numbers 1.2.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion offuran-2,4-dicarbaldehyde to 2-formylfuran-4-carboxylate. In oneembodiment, the dehydrogenase is classified as EC number 1.2.1. In oneembodiment, the dehydrogenase EC number 1.2.1 selected from aldehydedehydrogenase (NAD+) (EC number 1.2.1.3) or aldehyde dehydrogenase(NADP+) (EC number 1.2.1.4) or aldehyde dehydrogenase [NAD(P)+] (ECnumber 1.2.1.5) or 4-(γ-glutamylamino)butanal dehydrogenase (EC number1.2.1.99). In one embodiment, the oxidase is classified as EC number1.1.3. In one embodiment, the oxidase EC number 1.1.3 is5-(hydroxymethylfurfural oxidase (EC number 1.1.3.47). In someembodiments the EC 1.1.3.47 oxidase can be derived from the gene hmfH.In some embodiments, hmfH can be derived from Methylovorus sp. MP688 orCupriavidus basilensis. In one embodiment, the oxidase EC number 1.1.3is aryl-alcohol oxidase (EC number 1.1.3.7). In one embodiment, theperoxygenase is classified as EC number 1.11.2. In one embodiment, theperoxygenase EC number 1.11.2 is unspecific peroxygenase (EC number1.11.2.1).

In some aspects, 2-formylfuran-4-carboxylate is produced enzymatically,in the absence of microbes. In some aspects, 2-formylfuran-4-carboxylateis produced enzymatically in one or more vessels. In some aspects, theone or more vessels are substantially free of microbes. In some aspects,the enzymatic production of 2-formylfuran-4-carboxylate is performed inthe same step-wise fashion as described with in the methods utilizingrecombinant microorganisms, but substantially free of microorganisms orin the absence of microorganisms. In some aspects, the enzymes utilizedin the enzymatic production of 2-formylfuran-4-carboxylate are isolatedfrom microbes, recombinant or otherwise, and provided to theircorresponding substrates for the stepwise production of theintermediates utilized to produce 2-formylfuran-4-carboxylate. In someaspects, one or more of the steps of the methods are performed in thesame vessel. In some aspects, once the desired product is produced as aresult of the individual method steps described herein, the product isisolated and purified and then utilized as the substrate in the nextstep of the method of producing 2-formylfuran-4-carboxylate.

4-formylfuran-2-carboxylate

In one embodiment, the present disclosure provides a recombinantmicroorganism capable of producing 4-formylfuran-2-carboxylate from acarbon source. Some embodiments of the present disclosure are presentedin FIG. 1 , FIG. 2 , and FIG. 3 , which collectively detail thebiosynthetic conversion of a carbon feedstock to4-formylfuran-2-carboxylate.

In one embodiment, step G in FIG. 2 is a single step reaction utilizingfuran-2,4-dicarbaldehyde as a substrate. In one embodiment, thebioproduction of 4-formylfuran-2-carboxylate fromfuran-2,4-dicarbaldehyde is catalyzed by one or more enzymes representedby EC numbers 1.2.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion offuran-2,4-dicarbaldehyde to 4-formylfuran-2-carboxylate. In oneembodiment, the dehydrogenase is classified as EC number 1.2.1. In oneembodiment, the dehydrogenase EC number 1.2.1 selected from aldehydedehydrogenase (NAD+) (EC number 1.2.1.3) or aldehyde dehydrogenase(NADP+) (EC number 1.2.1.4) or aldehyde dehydrogenase [NAD(P)+] (ECnumber 1.2.1.5) or 4-(γ-glutamylamino)butanal dehydrogenase (EC number1.2.1.99). In one embodiment, the oxidase is classified as EC number1.1.3. In one embodiment, the oxidase EC number 1.1.3 is5-(hydroxymethylfurfural oxidase (EC number 1.1.3.47). In someembodiments the EC 1.1.3.47 oxidase can be derived from the gene hmfH.In some embodiments, hmfH can be derived from Methylovorus sp. MP688 orCupriavidus basilensis. In one embodiment, the oxidase EC number 1.1.3is aryl-alcohol oxidase (EC number 1.1.3.7). In one embodiment, theperoxygenase is classified as EC number 1.11.2. In one embodiment, theperoxygenase EC number 1.11.2 is unspecific peroxygenase (EC number1.11.2.1).

In one embodiment, step H in FIG. 2 is a single step reaction utilizing4-(hydroxymethyl)furoic acid as a substrate. In one embodiment, thebioproduction of 4-formylfuran-2-carboxylate from4-(hydroxymethyl)furoic acid is catalyzed by one or more enzymesrepresented by EC numbers 1.1.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion of4-(hydroxymethyl)furoic acid to 4-formylfuran-2-carboxylate. In oneembodiment, the dehydrogenase is classified as EC number 1.1.1. In oneembodiment, the dehydrogenase EC number 1.1.1 selected from alcoholdehydrogenase (EC number 1.1.1.1), or alcohol dehydrogenase (NADP+) (ECnumber 1.1.1.2), or D-xylose reductase (EC number 1.1.1.307), oraryl-alcohol dehydrogenase (EC number 1.1.1.90), or aryl-alcoholdehydrogenase (NADP+) (EC number 1.1.1.91). In one embodiment, theoxidase is classified as EC number 1.1.3. In one embodiment, the oxidaseEC number 1.1.3 is 5-(hydroxymethylfurfural oxidase (EC number1.1.3.47). In some embodiments the EC 1.1.3.47 oxidase can be derivedfrom the gene hmfH. In some embodiments, hmfH can be derived fromMethylovorus sp. MP688 or Cupriavidus basilensis. See Dijkman andFraaije (2014) and Koopman et al. (2010). In one embodiment, the oxidaseEC number 1.1.3 is aryl-alcohol oxidase (EC number 1.1.3.7). See Carroet al. (2015). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1). See Carro et al. (2015).

In some aspects, 4-formylfuran-2-carboxylate is produced enzymatically,in the absence of microbes. In some aspects, 4-formylfuran-2-carboxylateis produced enzymatically in one or more vessels. In some aspects, theone or more vessels are substantially free of microbes. In some aspects,the enzymatic production of 4-formylfuran-2-carboxylate is performed inthe same step-wise fashion as described with in the methods utilizingrecombinant microorganisms, but substantially free of microorganisms orin the absence of microorganisms. In some aspects, the enzymes utilizedin the enzymatic production of 4-formylfuran-2-carboxylate are isolatedfrom microbes, recombinant or otherwise, and provided to theircorresponding substrates for the stepwise production of theintermediates utilized to produce 4-formylfuran-2-carboxylate. In someaspects, one or more of the steps of the methods are performed in thesame vessel. In some aspects, once the desired product is produced as aresult of the individual method steps described herein, the product isisolated and purified and then utilized as the substrate in the nextstep of the method of producing 4-formylfuran-2-carboxylate.

Generation of Microbial Populations

Genetic Modification

The genetic modification introduced into one or more microbes of thepresent disclosure may alter or abolish a regulatory sequence of atarget gene. In some aspects, the genetic modification introduced intoone or more microbes of the present disclosure may introduce a new traitor phenotype into the one or more microbes. One or more regulatorysequences may also be inserted, including heterologous regulatorysequences and regulatory sequences found within a genome of an animal,plant, fungus, yeast, bacteria, or virus corresponding to the microbeinto which the genetic variation is introduced. Moreover, regulatorysequences may be selected based on the expression level of a gene in amicrobial culture. The genetic variation may be a pre-determined geneticvariation that is specifically introduced to a target site. In someaspects the genetic variation is a nucleic acid sequence that isintroduced into one or more microbial chromosomes. In some aspects, thegenetic variation is a nucleic acid sequence that is introduced into oneor more extrachromosomal nucleic acid sequence. The genetic variationmay be a random mutation within the target site. The genetic variationmay be an insertion or deletion of one or more nucleotides. In somecases, a plurality of different genetic variations (e.g. 2, 3, 4, 5, 10,or more) are introduced into one or more of the isolated bacteria. Theplurality of genetic variations can be any of the above types, the sameor different types, and in any combination. In some cases, a pluralityof different genetic variations are introduced serially, introducing afirst genetic variation after a first isolation step, a second geneticvariation after a second isolation step, and so forth so as toaccumulate a plurality of desired modifications in the microbes.

In some aspects, one or more of the substrates set forth in theproduction of the 2,4-FDCA monomers and polymers are biosynthesized froma carbon feedstock (e.g., glucose or glycerol).

In general, the term “genetic variation” refers to any change introducedinto a polynucleotide sequence relative to a reference polynucleotide,such as a reference genome or portion thereof, or reference gene orportion thereof. A genetic variation may be referred to as a “mutation,”and a sequence or organism comprising a genetic variation may bereferred to as a “genetic variant” or “mutant”. Genetic variations canhave any number of effects, such as the increase or decrease of somebiological activity, including gene expression, metabolism, and cellsignaling.

Genetic variations can be specifically introduced to a target site, orintroduced randomly. A variety of molecular tools and methods areavailable for introducing genetic variation. For example, geneticvariation can be introduced via polymerase chain reaction mutagenesis,oligonucleotide-directed mutagenesis, saturation mutagenesis, fragmentshuffling mutagenesis, homologous recombination, recombineering, lambdared mediated recombination, CRISPR/Cas9 systems, chemical mutagenesis,and combinations thereof. Chemical methods of introducing geneticvariation include exposure of DNA to a chemical mutagen, e.g., ethylmethanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitrosourea (ENU), N-methyl-N-nitro-N′-nitrosoguanidine, 4-nitroquinoline N-oxide,diethyl sulfate, benzopyrene, cyclophosphamide, bleomycin,triethylmelamine, acrylamide monomer, nitrogen mustard, vincristine,diepoxyalkanes (for example, diepoxybutane), ICR-170, formaldehyde,procarbazine hydrochloride, ethylene oxide, dimethylnitrosamine, 7,12dimethylbenz(a)anthracene, chlorambucil, hexamethylphosphoramide,bisulfan, and the like. Radiation mutation-inducing agents includeultraviolet radiation, γ-irradiation, X-rays, and fast neutronbombardment. Genetic variation can also be introduced into a nucleicacid using, e.g., trimethylpsoralen with ultraviolet light. Random ortargeted insertion of a mobile DNA element, e.g., a transposableelement, is another suitable method for generating genetic variation.

Genetic variations can be introduced into a nucleic acid duringamplification in a cell-free in vitro system, e.g., using a polymerasechain reaction (PCR) technique such as error-prone PCR. Geneticvariations can be introduced into a nucleic acid in vitro using DNAshuffling techniques (e.g., exon shuffling, domain swapping, and thelike). Genetic variations can also be introduced into a nucleic acid asa result of a deficiency in a DNA repair enzyme in a cell, e.g., thepresence in a cell of a mutant gene encoding a mutant DNA repair enzymeis expected to generate a high frequency of mutations (i.e., about 1mutation/100 genes-1 mutation/10,000 genes) in the genome of the cell.Examples of genes encoding DNA repair enzymes include but are notlimited to Mut H, Mut S, Mut L, and Mut U, and the homologs thereof inother species (e.g., MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1, and thelike). Example descriptions of various methods for introducing geneticvariations are provided in e.g., Stemple (2004) Nature 5:1-7; Chiang etal. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994) Proc. Natl.Acad. Sci. USA 91:10747-10751; and U.S. Pat. Nos. 6,033,861, and6,773,900.

Genetic variations introduced into microbes may be classified astransgenic, cisgenic, intragenomic, intrageneric, intergeneric,synthetic, evolved, rearranged, or SNPs.

CRISPR/Cas9 (Clustered regularly interspaced short palindromicrepeats)/CRISPR-associated (Cas) systems can be used to introducedesired mutations. CRISPR/Cas9 provide bacteria and archaea withadaptive immunity against viruses and plasmids by using CRISPR RNAs(crRNAs) to guide the silencing of invading nucleic acids. The Cas9protein (or functional equivalent and/or variant thereof, i.e.,Cas9-like protein) naturally contains DNA endonuclease activity thatdepends on the association of the protein with two naturally occurringor synthetic RNA molecules called crRNA and tracrRNA (also called guideRNAs). In some cases, the two molecules are covalently link to form asingle molecule (also called a single guide RNA (“sgRNA”). Thus, theCas9 or Cas9-like protein associates with a DNA-targeting RNA (whichterm encompasses both the two-molecule guide RNA configuration and thesingle-molecule guide RNA configuration), which activates the Cas9 orCas9-like protein and guides the protein to a target nucleic acidsequence. If the Cas9 or Cas9-like protein retains its natural enzymaticfunction, it will cleave target DNA to create a double-stranded break,which can lead to genome alteration (i.e., editing: deletion, insertion(when a donor polynucleotide is present), replacement, etc.), therebyaltering gene expression. Some variants of Cas9 (which variants areencompassed by the term Cas9-like) have been altered such that they havea decreased DNA cleaving activity (in some cases, they cleave a singlestrand instead of both strands of the target DNA, while in other cases,they have severely reduced to no DNA cleavage activity). Furtherexemplary descriptions of CRISPR systems for introducing geneticvariation can be found in, e.g. U.S. Pat. No. 8,795,965.

Oligonucleotide-directed mutagenesis, also called site-directedmutagenesis, typically utilizes a synthetic DNA primer. This syntheticprimer contains the desired mutation and is complementary to thetemplate DNA around the mutation site so that it can hybridize with theDNA in the gene of interest. The mutation may be a single base change (apoint mutation), multiple base changes, deletion, or insertion, or acombination of these. The single-strand primer is then extended using aDNA polymerase, which copies the rest of the gene. The gene thus copiedcontains the mutated site, and may then be introduced into a host cellas a vector and cloned. Finally, mutants can be selected by DNAsequencing to check that they contain the desired mutation.

Genetic variations can be introduced using error-prone PCR. In thistechnique, the gene of interest is amplified using a DNA polymeraseunder conditions that are deficient in the fidelity of replication ofsequence. The result is that the amplification products contain at leastone error in the sequence. When a gene is amplified and the resultingproduct(s) of the reaction contain one or more alterations in sequencewhen compared to the template molecule, the resulting products aremutagenized as compared to the template. Another means of introducingrandom mutations is exposing cells to a chemical mutagen, such asnitrosoguanidine or ethyl methanesulfonate (Nestmann, Mutat Res 1975June; 28(3):323-30), and the vector containing the gene is then isolatedfrom the host.

Homologous recombination mutagenesis involves recombination between anexogenous DNA fragment and the targeted polynucleotide sequence. After adouble-stranded break occurs, sections of DNA around the 5′ ends of thebreak are cut away in a process called resection. In the strand invasionstep that follows, an overhanging 3′ end of the broken DNA molecule then“invades” a similar or identical DNA molecule that is not broken. Themethod can be used to delete a gene, remove exons, add a gene, andintroduce point mutations. Homologous recombination mutagenesis can bepermanent or conditional. Typically, a recombination template is alsoprovided. A recombination template may be a component of another vector,contained in a separate vector, or provided as a separatepolynucleotide. In some aspects, a recombination template is designed toserve as a template in homologous recombination, such as within or neara target sequence nicked or cleaved by a site-specific nuclease. Atemplate polynucleotide may be of any suitable length, such as about ormore than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, ormore nucleotides in length. In some aspects, the template polynucleotideis complementary to a portion of a polynucleotide comprising the targetsequence. When optimally aligned, a template polynucleotide mightoverlap with one or more nucleotides of a target sequences (e.g. aboutor more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 100 or more nucleotides). In some aspects, when a template sequenceand a polynucleotide comprising a target sequence are optimally aligned,the nearest nucleotide of the template polynucleotide is within about 1,5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000,or more nucleotides from the target sequence. Non-limiting examples ofsite-directed nucleases useful in methods of homologous recombinationinclude zinc finger nucleases, CRISPR nucleases, TALE nucleases, andmeganuclease. For a further description of the use of such nucleases,see e.g. U.S. Pat. No. 8,795,965 and US20140301990.

Introducing genetic variation may be an incomplete process, such thatsome bacteria in a treated population of bacteria carry a desiredmutation while others do not. In some cases, it is desirable to apply aselection pressure so as to enrich for bacteria carrying a desiredgenetic variation. Traditionally, selection for successful geneticvariants involved selection for or against some functionality impartedor abolished by the genetic variation, such as in the case of insertingantibiotic resistance gene or abolishing a metabolic activity capable ofconverting a non-lethal compound into a lethal metabolite. It is alsopossible to apply a selection pressure based on a polynucleotidesequence itself, such that only a desired genetic variation need beintroduced (e.g. without also requiring a selectable marker). In thiscase, the selection pressure can comprise cleaving genomes lacking thegenetic variation introduced to a target site, such that selection iseffectively directed against the reference sequence into which thegenetic variation is sought to be introduced. Typically, cleavage occurswithin 100 nucleotides of the target site (e.g. within 75, 50, 25, 10,or fewer nucleotides from the target site, including cleavage at orwithin the target site). Cleaving may be directed by a site-specificnuclease selected from the group consisting of a Zinc Finger nuclease, aCRISPR nuclease, a TALE nuclease (TALEN), or a meganuclease. Such aprocess is similar to processes for enhancing homologous recombinationat a target site, except that no template for homologous recombinationis provided. As a result, bacteria lacking the desired genetic variationare more likely to undergo cleavage that, left unrepaired, results incell death. Bacteria surviving selection may then be isolated forassessing conferral of an improved trait.

A CRISPR nuclease may be used as the site-specific nuclease to directcleavage to a target site. An improved selection of mutated microbes canbe obtained by using Cas9 to kill non-mutated cells. Microbes can thenbe re-isolated from tissues. CRISPR nuclease systems employed forselection against non-variants can employ similar elements to thosedescribed above with respect to introducing genetic variation, exceptthat no template for homologous recombination is provided. Cleavagedirected to the target site thus enhances death of affected cells.

Other options for specifically inducing cleavage at a target site areavailable, such as zinc finger nucleases, TALE nuclease (TALEN) systems,and meganuclease. Zinc-finger nucleases (ZFNs) are artificial DNAendonucleases generated by fusing a zinc finger DNA binding domain to aDNA cleavage domain. ZFNs can be engineered to target desired DNAsequences and this enables zinc-finger nucleases to cleave unique targetsequences. When introduced into a cell, ZFNs can be used to edit targetDNA in the cell (e.g., the cell's genome) by inducing double strandedbreaks. Transcription activator-like effector nucleases (TALENs) areartificial DNA endonucleases generated by fusing a TAL (Transcriptionactivator-like) effector DNA binding domain to a DNA cleavage domain.TALENS can be quickly engineered to bind practically any desired DNAsequence and when introduced into a cell, TALENs can be used to edittarget DNA in the cell (e.g., the cell's genome) by inducing doublestrand breaks. Meganucleases (homing endonuclease) areendodeoxyribonucleases characterized by a large recognition site(double-stranded DNA sequences of 12 to 40 base pairs. Meganucleases canbe used to replace, eliminate or modify sequences in a highly targetedway. By modifying their recognition sequence through proteinengineering, the targeted sequence can be changed. Meganucleases can beused to modify all genome types, whether bacterial, plant or animal andare commonly grouped into four families: the LAGLIDADG family, theGIY-YIG family, the His-Cyst box family and the HNH family. Exemplaryhoming endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV,I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII andI-TevIII.

Microbes

As described herein, in some aspects, recombinant microorganisms arecapable of producing 4-HMF, 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, or 2,4-FDCA,and any combination thereof.

As described herein, in some aspects, the recombinant microorganisms areprokaryotic microorganism. In some aspects, the prokaryoticmicroorganisms are bacteria. “Bacteria”, or “eubacteria”, refers to adomain of prokaryotic organisms. Bacteria include at least elevendistinct groups as follows: (1) Gram-positive (gram+) bacteria, of whichthere are two major subdivisions: (1) high G+C group (Actinomycetes,Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus,Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas);(2) Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of gram positive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

In some aspects, the microorganisms of the present disclosure are fungi.

In some aspects, the recombinant microorganism is a eukaryoticmicroorganism. In some aspectsts, the eukaryotic microorganism is ayeast. In exemplary aspects, the yeast is a member of a genus selectedfrom the group consisting of Yarrowia, Candida, Saccharomyces, Pichia,Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces,Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon,Rhodotorula, and Myxozyma.

In some aspects, the recombinant microorganism is a prokaryoticmicroorganism. In exemplary aspects, the prokaryotic microorganism is amember of a genus selected from the group consisting of Escherichia,Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus,Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus,Arthrobacter, Corynebacterium, and Brevibacterium.

In some aspects, microorganism for use in the methods of the presentdisclosure can be selected from the group consisting of Yarrowia,Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia,Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen,Cryptococcus, Trichosporon, Rhodotorula, Myxozyma, Escherichia,Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus,Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus,Arthrobacter, Corynebacterium, and Brevibacterium.

In some aspects, a microbe resulting from the methods described hereinmay be a species selected from any of the following genera: Neisseria,Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus,Bordetella, Candida, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, Fusobacterium, Actinomyces, Bacillus,Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria,Mycobacterium, Myxococcus, Nocardia, Issatchenkia, Staphylococcus,Streptococcus, Streptomyces, Saccharomyces, Pichia, and Aspergillus.

In some aspects, microorganisms for use in the methods of the presentdisclosure include Clostridium sp., Clostridium ljungdahlii, Clostridiumautoethanogenum, Clostridium ragsdalei, Eubacterium limosum,Butyribacterium methylotrophicum, Moorella thermoacetica,Corynebacterium glutamicum, Clostridium aceticum, Acetobacterium woodii,Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans,Clostridium formicoaceticum, Clostridium scatologenes, Moorellathermoautotrophica, Acetonema longum, Blautia producta, Clostridiumglycolicum, Clostridium magnum, Candida krusei, Clostridium mayombei,Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridiumbeijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusaovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum,Issatchenkia orientalis, Sporomusa termitida, Moorella glycerini,Eubacterium aggregans, Treponema azotonutricium, Pichia kudriavzevii,Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillussp, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis,and Terrisporobacter glycolicus.

The term “recombinant microorganism” and “recombinant host cell” areused interchangeably herein and refer to microorganisms that have beengenetically modified to express or to overexpress endogenous enzymes, toexpress heterologous enzymes, such as those included in a vector, in anintegration construct, or which have an alteration in expression of anendogenous gene. By “alteration” it is meant that the expression of thegene, or level of a RNA molecule or equivalent RNA molecules encodingone or more polypeptides or polypeptide subunits, or activity of one ormore polypeptides or polypeptide subunits is up regulated or downregulated, such that expression, level, or activity is greater than orless than that observed in the absence of the alteration. For example,the term “alter” can mean “inhibit,” but the use of the word “alter” isnot limited to this definition. It is understood that the terms“recombinant microorganism” and “recombinant host cell” refer not onlyto the particular recombinant microorganism but to the progeny orpotential progeny of such a microorganism. Because certain modificationsmay occur in succeeding generations due to either mutation orenvironmental influences, such progeny may not, in fact, be identical tothe parent cell, but are still included within the scope of the term asused herein.

Culturing of the microorganisms used in the methods of the disclosuremay be conducted using any number of processes known in the art forculturing and fermenting substrates using the microorganisms of thepresent disclosure.

The fermentation may be carried out in any suitable bioreactor, such asContinuous Stirred Tank Bioreactor, Bubble Column Bioreactor, AirliftBioreactor, Fluidized Bed Bioreactor, Packed Bed Bioreactor,Photo-Bioreactor, Immobilized Cell Reactor, Trickle Bed Reactor, MovingBed Biofilm Reactor, Bubble Column, Gas Lift Fermenter, MembraneReactors such as Hollow Fiber Membrane Bioreactor. In some aspects, thebioreactor comprises a first, growth reactor in which the microorganismsare cultured, and a second, fermentation reactor, to which fermentationbroth from the growth reactor is fed and in which most of thefermentation product is produced. In some aspects, the bioreactorsimultaneously accomplishes the culturing of microorganism and theproducing the fermentation product from carbon sources such substratesand/or feedstocks provided.

In some aspects, the disclosure is drawn to a method ofrecovering/isolating a 2,4-FDCA monomer. In some aspects, the disclosureis drawn to a method of recovering/isolating 4-HMF, 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, or 2,4-FDCA,and any combination thereof. In some aspects, the disclosure is drawn toa method of recovering/isolating a 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, and/or 4-formylfuran-2-carboxylate monomeror polymer. In some aspects, the disclosure is drawn to a method ofrecovering/isolating 4-HMF, 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, or 2,4-FDCA,and any combination thereof. The recovery/collection/isolation can be bymethods known in the art, such as distillation, membrane-basedseparation gas stripping, precipitation, solvent extraction, andexpanded bed adsorption.

Feedstock

In some aspects, the feedstock comprises a carbon source. In someaspects, the carbon source may be selected from sugars, glycerol,alcohols, organic acids, alkanes, fatty acids, lignocellulose, proteins,carbon dioxide, and carbon monoxide. In one aspect, the carbon source isa sugar. In one aspect, the sugar is a monosaccharide. In one aspect,the sugar is a polysaccharide. In one aspect, the sugar is glucose oroligomers of glucose thereof. In one aspect, the oligomers of glucoseare selected from fructose, sucrose, starch, cellobiose, maltose,lactose and cellulose. In one aspect, the sugar is a five carbon sugar.In one aspect, the sugar is a six carbon sugar. In some aspects, thefeedstock comprises one or more five carbon sugars and/or one or moresix carbon sugars. In some aspects, the feedstock comprises one or moreof xylose, glucose, arabinose, galactose, maltose, fructose, mannose,sucrose, and/or combinations thereof. In some aspects, the feedstockcomprises one or more of xylose and/or glucose. In some aspects, thefeedstock comprises one or more of arabinose, galactose, maltose,fructose, mannose, sucrose, and/or combinations thereof.

In some aspects, the microbes utilize one or more five carbon sugars(pentoses) and/or one or more six carbon sugars (hexoses). In someaspects, the microbes utilize one or more of xylose and/or glucose. Insome aspects, the microbes utilize one or more of arabinose, galactose,maltose, fructose, mannose, sucrose, and/or combinations thereof. Insome aspects, the microbes utilize one or more of xylose, glucose,arabinose, galactose, maltose, fructose, mannose, sucrose, and/orcombinations thereof.

In some aspects, hexoses may be selected from D-allose, D-altrose,D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose,D-tagtose, D-sorbose, D-fructose, D-psicose, and other hexoses known inthe art. In some aspects, pentoses may be selected from D-xylose,D-ribose, D-arabinose, D-lyxose, D-xylulose, D-ribulose, and otherpentoses known in the art. In some aspects, the hexoses and pentoses maybe selected from the levorotary or dextrorotary enantiomer of any of thehexoses and pentoses disclosed herein.

Microbial Compositions

In some aspects, the microbes of the disclosure are combined intomicrobial compositions.

In some aspects, the microbial compositions of the present disclosureare solid. Where solid compositions are used, it may be desired toinclude one or more carrier materials including, but not limited to:mineral earths such as silicas, talc, kaolin, limestone, chalk, clay,dolomite, diatomaceous earth; calcium sulfate; magnesium sulfate;magnesium oxide; zeolites, calcium carbonate; magnesium carbonate;trehalose; chitosan; shellac; albumins; starch; skim milk powder; sweetwhey powder; maltodextrin; lactose; inulin; dextrose; and products ofvegetable origin such as cereal meals, tree bark meal, wood meal, andnutshell meal.

In some aspects, the microbial compositions of the present disclosureare liquid. In further aspects, the liquid comprises a solvent that mayinclude water or an alcohol or a saline or carbohydrate solution. Insome aspects, the microbial compositions of the present disclosureinclude binders such as polymers, carboxymethylcellulose, starch,polyvinyl alcohol, and the like.

In some aspects, microbial compositions of the present disclosurecomprise saccharides (e.g., monosaccharides, disaccharides,trisaccharides, polysaccharides, oligosaccharides, and the like),polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides,proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acidpolymers, silica, inorganic salts and combinations thereof. In furtheraspect, microbial compositions comprise polymers of agar, agarose,gelrite, gellan gum, and the like. In some aspects, microbialcompositions comprise plastic capsules, emulsions (e.g., water and oil),membranes, and artificial membranes. In some aspects, emulsions orlinked polymer solutions comprise microbial compositions of the presentdisclosure. See Harel and Bennett (U.S. Pat. No. 8,460,726 B2).

In some aspects, microbial compositions of the present disclosure occurin a solid form (e.g., dispersed lyophilized spores) or a liquid form(microbes interspersed in a storage medium). In some aspects, microbialcompositions of the present disclosure are added in dry form to a liquidto form a suspension immediately prior to use.

Methods of Producing Biosynthesis Products

The present disclosure provides a method of producing one or morebiosynthesis products using a recombinant microorganisms. Thebiosynthesis products include: 4-HMF, 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and 2,4-FDCA.In one embodiment, the method comprises cultivating the recombinantmicroorganism in a culture medium. In one embodiment, the culture mediumcontains a feedstock comprising a carbon source that the recombinantmicroorganism can utilize to produce the one or more biosynthesisproducts. In one embodiment, the carbon source in the culture medium isselected from the group that comprises a hexose, a pentose, or glycerol.In certain embodiments, the carbon source is glycerol. Some embodimentsof the present disclosure are presented in FIG. 1 , FIG. 2 , and FIG. 3, which collectively detail the biosynthetic conversion of a carbonfeedstock to one or more of the biosynthesis products.

The present disclosure provides a method of producing a recombinantmicroorganism that produces 4-HMF, 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and 2,4-FDCAfrom a feedstock comprising an exogenous carbon source. In oneembodiment, the method comprises introducing into and/or overexpressingin the recombinant microorganism endogenous and/or exogenous nucleicacid molecules capable of converting a carbon source into 4-HMF,2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoicacid, 2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and2,4-FDCA. In one embodiment, the carbon source may include glyceroland/or monosaccharides.

In one embodiment, endogenous and/or exogenous nucleic acid moleculesconvert glycerol or a monosaccharide into glyceraldehyde 3-phosphate(G3P). G3P is a common natural intermediary metabolite. In someembodiments, it can be produced from glucose via the glycolysis pathwayor from xylose via the pentose phosphate pathway, or from glycerol. Inone embodiment, the recombinant microorganism capable of producing4-HMF, 2,4-furandimethanol, furan-2,4-dicarbaldehyde,4-(hydroxymethyl)furoic acid, 2-formylfuran-4-carboxylate,4-formylfuran-2-carboxylate, and 2,4-FDCA utilizes a carbon source thatcomprises a hexose, a pentose, or glycerol. In certain embodiments, thecarbon source is glycerol.

In one embodiment, the present disclosure contemplates methods ofproducing 2,4-FDCA and the multiple steps and processes for producing2,4-FDCA. In some embodiments, the present disclosure contemplates theindividual methods for producing one or more of 4-HMF,2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoicacid, 2-formylfuran-4-carboxylate, and 4-formylfuran-2-carboxylate, thatare described in the process of making 2,4-FDCA.

In one embodiment, the recombinant microorganisms of the method arederived from a parental microorganism selected from the group consistingof Clostridium sp., Clostridium ljungdahlii, Clostridiumautoethanogenum, Clostridium ragsdalei, Eubacterium limosum,Butyribacterium methylotrophicum, Moorella thermoacetica,Corynebacterium glutamicum, Clostridium aceticum, Acetobacterium woodii,Alkalibaculum bacchii, Clostridium drakei, Clostridium carboxidivorans,Clostridium formicoaceticum, Clostridium scatologenes, Moorellathermoautotrophica, Acetonema longum, Blautia producta, Clostridiumglycolicum, Clostridium magnum, Candida krusei, Clostridium mayombei,Clostridium methoxybenzovorans, Clostridium acetobutylicum, Clostridiumbeijerinckii, Oxobacter pfennigii, Thermoanaerobacter kivui, Sporomusaovata, Thermoacetogenium phaeum, Acetobacterium carbinolicum,Issatchenkia orientalis, Sporomusa termitida, Moorella glycerini,Eubacterium aggregans, Treponema azotonutricium, Pichia kudriavzevii,Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillussp, Corynebacterium sp., Yarrowia lipolytica, Scheffersomyces stipitis,and Terrisporobacter glycolicus.

4-HMF

In one embodiment, the present disclosure comprises converting one ormore carbon sources to glyceraldehyde 3-phosphate (G3P); converting G3Pto (5-formylfuran-3-yl)methyl phosphate (Step A); converting(5-formylfuran-3-yl)methyl phosphate to 4-hydroxymethylfurfural (4-HMF)(Step B).

In one embodiment, the disclosure is drawn to a method of producing arecombinant microorganism of any one of the embodiments disclosed hereincomprising an endogenous and/or exogenous nucleic acid molecules capableof converting a carbon source to glyceraldehyde 3-phosphate (G3P). Inone embodiment, glycerol is converted to glycerol-3-phopshate by atleast one endogenous or exogenous nucleic acid molecule encoding aglycerol kinase. In one embodiment, glycerol-3-phosphate is converted todihydroxyacetone phosphate (DHAP) by at least one endogenous orexogenous nucleic acid molecule encoding a glycerol-3-phosphatedehydrogenase. In one embodiment, glycerol is converted todihydroxyacetone by at least one endogenous or exogenous nucleic acidmolecule encoding a glycerol dehydrogenase. In one embodiment,dihydroxyacetone is converted to dihydroxyacetone phosphate (DHAP) by atleast one endogenous or exogenous nucleic acid molecule encoding adihydroxyacetone kinase. In one embodiment, DHAP is converted to G3P byat least one endogenous or exogenous nucleic acid molecule encoding atriose phosphate isomerase. See Zhang et al. (2010).

In one embodiment, the disclosure is drawn to a method of producing arecombinant microorganism of any one of the embodiments of disclosedherein comprising at least one endogenous or exogenous nucleic acidmolecule encoding a (5-formylfuran-3-yl)methyl phosphate synthase thatcatalyzes the conversion of G3P to (5-formylfuran-3-yl)methyl phosphate.In one embodiment, the (5-formylfuran-3-yl)methyl phosphate synthase isclassified as EC number 4.2.3.153. In some embodiments the EC 4.2.3.153(5-formylfuran-3-yl)methyl phosphate synthase can be derived from thegene mfnB. In some embodiments, mfnB can be derived fromMethanocaldococcus jannaschii. In some embodiments, the(5-formylfuran-3-yl)methyl phosphate synthase can be derived from enzymecandidates listed at Table 1. In some embodiments, the(5-formylfuran-3-yl)methyl phosphate synthase is homologous or similarto the enzymes listed at Table 1. In some embodiments, an(5-formylfuran-3-yl)methyl phosphate synthase enzyme is evolved orengineered to improve its catalytic efficiency, markedly kcat.

In one embodiment, the disclosure is drawn to a method of producing arecombinant microorganism of any one of the embodiments disclosed hereincomprising at least one endogenous or exogenous nucleic acid moleculeencoding a phosphatase that catalyzes the conversion of(5-formylfuran-3-yl)methyl phosphate to (4-HMF). In one embodiment, thephosphatase is classified as haloacid dehalogenase (Koonin et al. J.Mol. Biol. 244(1). 1994). In some aspects, the phosphatase of reaction bis endogenous to the host (Offley et al. Curr. Gen. 65. 2019). In someaspects, the phosphatase enzyme endogenous to the host is overexpressed.In some cases a heterologous phosphatase able to perform the desiredreaction is used and is selected from an alkaline phosphatase, acidphosphatase, fructose-bisphosphatase, sugar-phosphatase, orsugar-terminal-phosphatase. In some embodiments, the phosphatase can bederived from enzyme candidates listed at Table 2. In some embodiments,the phosphatase is homologous or similar to the enzymes listed at Table2. In some embodiments, an phosphatase enzyme is evolved or engineeredto improve its catalytic efficiency and or specificity for theconversion of (5-formylfuran-3-yl)methyl phosphate to (4-HMF).

Accordingly, in one embodiment, the disclosure is drawn to a method ofproducing a recombinant microorganism that comprises endogenous and/orexogenous nucleic acid molecules capable of converting a carbon sourceto glyceraldehyde 3-phosphate (G3P); at least one endogenous orexogenous nucleic acid molecule encoding a (5-formylfuran-3-yl)methylphosphate synthase that catalyzes the conversion of G3P to(5-formylfuran-3-yl)methyl phosphate; at least one endogenous orexogenous nucleic acid molecule encoding a phosphatase that catalyzesthe conversion of (5-formylfuran-3-yl)methyl phosphate to 4-HMF.

2,4-FDCA

In one embodiment, methods of the disclosure convert G3P to 2,4-FDCA viaseveral enzymatically-catalyzed successive steps. In one embodiment, thepresent disclosure comprises converting one or more carbon sources toglyceraldehyde 3-phosphate (G3P); converting G3P to(5-formylfuran-3-yl)methyl phosphate (Step A); converting(5-formylfuran-3-yl)methyl phosphate to 4-hydroxymethylfurfural (4-HMF)(Step B); converting 4-HMF to 2,4 FDCA directly (Step C) or or throughthe production of intermediates, as converting 4-HMF tofuran-2,4-dicarbaldehyde (Step D) and/or 4-(hydroxymethyl)furoic acid(Step E); converting furan-2,4-dicarbaldehyde to4-formylfuran-2-carboxylate (Step G) and/or 2-formylfuran-4-carboxylate(Step F) and/or converting 4-(hydroxymethyl)furoic acid to4-formylfuran-2-carboxylate (Step H); converting4-formylfuran-2-carboxylate to 2,4-FDCA (Step J) and/or converting2-formylfuran-4-carboxylate to 2,4-FDCA (Step I). In a furtherembodiment, the one or more carbon sources may include glycerol or amonosaccharide.

Accordingly, in one embodiment, provided herein is a method of producinga recombinant microorganism that comprises an endogenous and/orexogenous nucleic acid molecules capable of converting a carbon sourceto glyceraldehyde 3-phosphate (G3P); at least one endogenous orexogenous nucleic acid molecule encoding a (5-formylfuran-3-yl)methylphosphate synthase that catalyzes the conversion of G3P to(5-formylfuran-3-yl)methyl phosphate; at least one endogenous orexogenous nucleic acid molecule encoding a phosphatase that catalyzesthe conversion of (5-formylfuran-3-yl)methyl phosphate to (4-HMF); atleast one endogenous or exogenous nucleic acid molecule encoding adehydrogenase or an oxidase or a peroxygenase that catalyzes theconversion of 4-HMF to 2,4 FDCA directly or through the production ofintermediates, as furan-2,4-dicarbaldehyde and/or4-(hydroxymethyl)furoic acid; at least one endogenous or exogenousnucleic acid molecule encoding a dehydrogenase or an oxidase or aperoxygenase that catalyzes the conversion of furan-2,4-dicarbaldehydeto 4-formylfuran-2-carboxylate and/or 2-formylfuran-4-carboxylate and/orthe conversion of 4-(hydroxymethyl)furoic acid to4-formylfuran-2-carboxylate; at least one endogenous or exogenousnucleic acid molecule encoding a dehydrogenase or an oxidase or aperoxygenase that catalyzes the conversion of2-formylfuran-4-carboxylate to 2,4-FDCA and/or4-formylfuran-2-carboxylate to 2,4-FDCA.

In one embodiment, the recombinant microorganism of any one of theembodiments of the method disclosed herein comprise an endogenous and/orexogenous nucleic acid molecules capable of converting a carbon sourceto glyceraldehyde 3-phosphate (G3P). In one embodiment, glycerol isconverted to glycerol-3-phopshate by at least one endogenous orexogenous nucleic acid molecule encoding a glycerol kinase. In oneembodiment, glycerol-3-phosphate is converted to dihydroxyacetonephosphate (DHAP) by at least one endogenous or exogenous nucleic acidmolecule encoding a glycerol-3-phosphate dehydrogenase. In oneembodiment, glycerol is converted to dihydroxyacetone by at least oneendogenous or exogenous nucleic acid molecule encoding a glyceroldehydrogenase. In one embodiment, dihydroxyacetone is converted todihydroxyacetone phosphate (DHAP) by at least one endogenous orexogenous nucleic acid molecule encoding a dihydroxyacetone kinase. Inone embodiment, DHAP is converted to G3P by at least one endogenous orexogenous nucleic acid molecule encoding a triose phosphate isomerase.

In one embodiment, the recombinant microorganism of any one of theembodiments of the method disclosed herein comprise at least oneendogenous or exogenous nucleic acid molecule encoding a(5-formylfuran-3-yl)methyl phosphate synthase that catalyzes theconversion of G3P to (5-formylfuran-3-yl)methyl phosphate. In oneembodiment, the (5-formylfuran-3-yl)methyl phosphate synthase isclassified as EC number 4.2.3.153. In some embodiments the EC 4.2.3.153(5-formylfuran-3-yl)methyl phosphate synthase can be derived from thegene mfnB. In some embodiments, mfnB can be derived fromMethanocaldococcus jannaschii. In some embodiments, EC 4.2.3.153 can bederived from homologs of mfnB

In one embodiment, the recombinant microorganism of any one of theembodiments of the method disclosed herein comprise at least oneendogenous or exogenous nucleic acid molecule encoding a phosphatasethat catalyzes the conversion of (5-formylfuran-3-yl)methyl phosphate to(4-HMF). In one embodiment, the phosphatase is classified as EC number3.1.3. In one embodiment, the phosphatase EC number 3.1.3 phosphatase isselected from an alkaline phosphatase (EC number 3.1.3.1), acidphosphatase (EC number 3.1.3.2), fructose-bisphosphatase (EC number3.1.3.11), sugar-phosphatase (EC number 3.1.3.23), orsugar-terminal-phosphatase (EC number 3.1.3.58). In one embodiment, thekinase is classified as EC number 2.7.1. In one embodiment, the kinaseEC number 2.7.1 is selected from fructokinase (EC number 2.7.1.4),ribokinase (EC number 2.7.1.15), ribulokinase (EC number 2.7.1.16),xylulokinase (EC number 2.7.1.17), or D-ribulokinase (EC number2.7.1.47).

In one embodiment, the recombinant microorganism of any one of theembodiments of the method disclosed herein comprise at least oneendogenous or exogenous nucleic acid molecule encoding a dehydrogenase,or a oxidase, or a peroxygenase that catalyzes the conversion of 4-HMFto furan-2,4-dicarbaldehyde. In one embodiment, the dehydrogenase isclassified as EC number 1.1.1. In one embodiment, the dehydrogenase ECnumber 1.1.1 selected from alcohol dehydrogenase (EC number 1.1.1.1), oralcohol dehydrogenase (NADP+) (EC number 1.1.1.2), or D-xylose reductase(EC number 1.1.1.307), or aryl-alcohol dehydrogenase (EC number1.1.1.90), or aryl-alcohol dehydrogenase (NADP+) (EC number 1.1.1.91).In one embodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. In oneembodiment, the oxidase EC number 1.1.3 is aryl-alcohol oxidase (ECnumber 1.1.3.7). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1).

In one embodiment, the recombinant microorganism of any one of theembodiments of the method disclosed herein comprise at least oneendogenous or exogenous nucleic acid molecule encoding a dehydrogenase,or a oxidase, or a peroxygenase that catalyzes the conversion of 4-HMFto 4-(hydroxymethyl)furoic acid. In one embodiment, the dehydrogenase isclassified as EC number 1.2.1. In one embodiment, the dehydrogenase ECnumber 1.2.1 selected from aldehyde dehydrogenase (NAD+) (EC number1.2.1.3) or aldehyde dehydrogenase (NADP+) (EC number 1.2.1.4) oraldehyde dehydrogenase [NAD(P)+] (EC number 1.2.1.5) or4-(γ-glutamylamino)butanal dehydrogenase (EC number 1.2.1.99). In oneembodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. In oneembodiment, the oxidase EC number 1.1.3 is aryl-alcohol oxidase (ECnumber 1.1.3.7). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1).

In one embodiment, the recombinant microorganism of any one of theembodiments of the method disclosed herein comprise at least oneendogenous or exogenous nucleic acid molecule encoding a dehydrogenase,or a oxidase, or a peroxygenase that catalyzes the conversion offuran-2,4-dicarbaldehyde to 4-formylfuran-2-carboxylate and/or to2-formylfuran-4-carboxylate. In one embodiment, the dehydrogenase isclassified as EC number 1.2.1. In one embodiment, the dehydrogenase ECnumber 1.2.1 selected from aldehyde dehydrogenase (NAD+) (EC number1.2.1.3) or aldehyde dehydrogenase (NADP+) (EC number 1.2.1.4) oraldehyde dehydrogenase [NAD(P)+] (EC number 1.2.1.5) or4-(γ-glutamylamino)butanal dehydrogenase (EC number 1.2.1.99). In oneembodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. In oneembodiment, the oxidase EC number 1.1.3 is aryl-alcohol oxidase (ECnumber 1.1.3.7). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1).

In one embodiment, the recombinant microorganism of any one of theembodiments of the method disclosed herein comprise at least oneendogenous or exogenous nucleic acid molecule encoding a dehydrogenase,or a oxidase, or a peroxygenase that catalyzes the conversion of4-(hydroxymethyl)furoic acid to 4-formylfuran-2-carboxylate. In oneembodiment, the dehydrogenase is classified as EC number 1.1.1. In oneembodiment, the dehydrogenase EC number 1.1.1 selected from alcoholdehydrogenase (EC number 1.1.1.1), or alcohol dehydrogenase (NADP+) (ECnumber 1.1.1.2), or D-xylose reductase (EC number 1.1.1.307), oraryl-alcohol dehydrogenase (EC number 1.1.1.90), or aryl-alcoholdehydrogenase (NADP+) (EC number 1.1.1.91). In one embodiment, thedehydrogenase EC number 1.1.1 is. In one embodiment, the oxidase isclassified as EC number 1.1.3. In one embodiment, the oxidase EC number1.1.3 is 5-(hydroxymethylfurfural oxidase (EC number 1.1.3.47). In someembodiments the EC 1.1.3.47 oxidase can be derived from the gene hmfH.In some embodiments, hmfH can be derived from Methylovorus sp. MP688 orCupriavidus basilensis. In one embodiment, the oxidase EC number 1.1.3is aryl-alcohol oxidase (EC number 1.1.3.7). In one embodiment, theperoxygenase is classified as EC number 1.11.2. In one embodiment, theperoxygenase EC number 1.11.2 is unspecific peroxygenase (EC number1.11.2.1).

In one embodiment, the recombinant microorganism of any one of theembodiments of the method disclosed herein comprise at least oneendogenous or exogenous nucleic acid molecule encoding a dehydrogenase,or a oxidase, or a peroxygenase that catalyzes the conversion of4-formylfuran-2-carboxylate and/or 2-formylfuran-4-carboxylate to2,4-FDCA. In one embodiment, the dehydrogenase is classified as ECnumber 1.1.1. In one embodiment, the dehydrogenase EC number 1.1.1selected from alcohol dehydrogenase (EC number 1.1.1.1), or alcoholdehydrogenase (NADP+) (EC number 1.1.1.2), or D-xylose reductase (ECnumber 1.1.1.307), or aryl-alcohol dehydrogenase (EC number 1.1.1.90),or aryl-alcohol dehydrogenase (NADP+) (EC number 1.1.1.91). In oneembodiment the dehydrogenases can be derived from enzyme candidateslisted at Table 3. In some embodiments, the dehydrogenases arehomologous or similar to the enzymes listed at Table 3. In someembodiments, a dehydrogenases is evolved or engineered to improve itscatalytic efficiency against its desirable substrate.

In one embodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the HMF oxidase can bederived from the gene hmfH. In some embodiments, HMF oxidase can bederived from Methylovorus sp. MP688 or Cupriavidus basilensis. SeeDijkman and Fraaije (2014. Applied Environmental Microbiology,80.3:1082-1090) and Koopman et al. (2010. PNAS, 107(11):4919-4924). Inone embodiment, the HMF oxidase EC number 1.1.3 is aryl-alcohol oxidase(EC number 1.1.3.7). See Carro et al. (2015). In one embodiment, theperoxygenase is classified as EC number 1.11.2. In one embodiment, theperoxygenase EC number 1.11.2 is unspecific peroxygenase (EC number1.11.2.1). See Carro et al. (2015). In some embodiments, the HMF oxidasecan be derived from enzyme candidates listed at Table 4. In someembodiments, the HMF oxidase is homologous or similar to the enzymeslisted at Table 4. In some embodiments, the HMF oxidase enzyme isevolved or engineered to improve its catalytic efficiency.

2,4-furandimethanol

In one embodiment, the present disclosure is drawn to a method ofproducing a recombinant microorganism capable of producing2,4-furandimethanol from a carbon source. Some embodiments of thepresent disclosure are presented in FIG. 1 , FIG. 2 , and FIG. 3 , whichcollectively detail the biosynthetic conversion of a carbon feedstock to2,4-furandimethanol.

In one embodiment, the bioproduction of 2,4-furandimethanol from 4-HMFis catalyzed by a dehydrogenase encoded by the microorganism. In oneembodiment, the dehydrogenase is classified as EC number 1.1.1. In oneembodiment, the dehydrogenase EC number 1.1.1 is selected from alcoholdehydrogenase (EC number 1.1.1.1). In one embodiment, the dehydrogenaseEC number 1.1.1 is selected from alcohol dehydrogenase (NADP+) (ECnumber 1.1.1.2). In one embodiment, the dehydrogenase EC number 1.1.1 isselected from D-xylose reductase (EC number 1.1.1.90). In oneembodiment, the dehydrogenase EC number 1.1.1 is selected fromaryl-alcohol dehydrogenase (EC number 1.1.1.91). In one embodiment thedehydrogenases can be derived from enzyme candidates listed at Table 5.In some embodiments, the dehydrogenases are homologous or similar to theenzymes listed at Table 5. In some embodiments, a dehydrogenases isevolved or engineered to improve its catalytic efficiency for 4-HMFreduction to 2,4-furandimethanol.

In some aspects, 2,4-furandimethanol is produced enzymatically, in theabsence of microbes. In some aspects, 2,4-furandimethanol is producedenzymatically in one or more vessels. In some aspects, the one or morevessels are substantially free of microbes. In some aspects, theenzymatic production of 2,4-furandimethanol is performed in the samestep-wise fashion as described with in the methods utilizing recombinantmicroorganisms, but substantially free of microorganisms or in theabsence of microorganisms. In some aspects, the enzymes utilized in theenzymatic production of 2,4-furandimethanol are isolated from microbes,recombinant or otherwise, and provided to their corresponding substratesfor the stepwise production of the intermediates utilized to produce2,4-furandimethanol. In some aspects, one or more of the steps of themethods are performed in the same vessel. In some aspects, once thedesired product is produced as a result of the individual method stepsdescribed herein, the product is isolated and purified and then utilizedas the substrate in the next step of the method of producing2,4-furandimethanol.

Furan-2,4-dicarbaldehyde

In one embodiment, the present disclosure is drawn to a method ofproducing a recombinant microorganism capable of producingfuran-2,4-dicarbaldehyde from a carbon source. Some embodiments of thepresent disclosure are presented in FIG. 1 , FIG. 2 , and FIG. 3 , whichcollectively detail the biosynthetic conversion of a carbon feedstock tofuran-2,4-dicarbaldehyde.

In one embodiment, step D in FIG. 2 is a single step reaction utilizing4-HMF as a substrate. In one embodiment, the bioproduction offuran-2,4-dicarbaldehyde from 4-HMF is catalyzed by one or more enzymesrepresented by EC numbers 1.1.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, an oxidase, ora peroxygenase that catalyzes the conversion of 4-HMF tofuran-2,4-dicarbaldehyde. In one embodiment, the dehydrogenase isclassified as EC number 1.1.1. In one embodiment, the dehydrogenase ECnumber 1.1.1 selected from alcohol dehydrogenase (EC number 1.1.1.1), oralcohol dehydrogenase (NADP+) (EC number 1.1.1.2), or D-xylose reductase(EC number 1.1.1.307), or aryl-alcohol dehydrogenase (EC number1.1.1.90), or aryl-alcohol dehydrogenase (NADP+) (EC number 1.1.1.91).In one embodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. SeeDijkman and Fraaije (2014) and Koopman et al. (2010). In one embodiment,the oxidase EC number 1.1.3 is aryl-alcohol oxidase (EC number 1.1.3.7).See Carro et al. (2015). In one embodiment, the peroxygenase isclassified as EC number 1.11.2. In one embodiment, the peroxygenase ECnumber 1.11.2 is unspecific peroxygenase (EC number 1.11.2.1). See Carroet al. (2015).

In some aspects, furan-2,4-dicarbaldehyde is produced enzymatically, inthe absence of microbes. In some aspects, furan-2,4-dicarbaldehyde isproduced enzymatically in one or more vessels. In some aspects, the oneor more vessels are substantially free of microbes. In some aspects, theenzymatic production of furan-2,4-dicarbaldehyde is performed in thesame step-wise fashion as described with in the methods utilizingrecombinant microorganisms, but substantially free of microorganisms orin the absence of microorganisms. In some aspects, the enzymes utilizedin the enzymatic production of furan-2,4-dicarbaldehyde are isolatedfrom microbes, recombinant or otherwise, and provided to theircorresponding substrates for the stepwise production of theintermediates utilized to produce furan-2,4-dicarbaldehyde. In someaspects, one or more of the steps of the methods are performed in thesame vessel. In some aspects, once the desired product is produced as aresult of the individual method steps described herein, the product isisolated and purified and then utilized as the substrate in the nextstep of the method of producing furan-2,4-dicarbaldehyde.

4-(hydroxymethyl)furoic acid

In one embodiment, the present disclosure is drawn to a method ofproducing a recombinant microorganism capable of producing4-(hydroxymethyl)furoic acid from a carbon source. Some embodiments ofthe present disclosure are presented in FIG. 1 , FIG. 2 , and FIG. 3 ,which collectively detail the biosynthetic conversion of a carbonfeedstock to 4-(hydroxymethyl)furoic acid.

In one embodiment, step E in FIG. 2 is a single step reaction utilizing4-HMF as a substrate. In one embodiment, the bioproduction of4-(hydroxymethyl)furoic acid from 4-HMF is catalyzed by one or moreenzymes represented by EC numbers 1.1.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion of 4-HMF to4-(hydroxymethyl)furoic acid. In one embodiment, the dehydrogenase isclassified as EC number 1.2.1. In one embodiment, the dehydrogenase ECnumber 1.2.1 selected from aldehyde dehydrogenase (NAD+) (EC number1.2.1.3) or aldehyde dehydrogenase (NADP+) (EC number 1.2.1.4) oraldehyde dehydrogenase [NAD(P)+] (EC number 1.2.1.5) or4-(γ-glutamylamino)butanal dehydrogenase (EC number 1.2.1.99). In oneembodiment, the oxidase is classified as EC number 1.1.3. In oneembodiment, the oxidase EC number 1.1.3 is 5-(hydroxymethylfurfuraloxidase (EC number 1.1.3.47). In some embodiments the EC 1.1.3.47oxidase can be derived from the gene hmfH. In some embodiments, hmfH canbe derived from Methylovorus sp. MP688 or Cupriavidus basilensis. In oneembodiment, the oxidase EC number 1.1.3 is aryl-alcohol oxidase (ECnumber 1.1.3.7). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1).

In some aspects, 4-(hydroxymethyl)furoic acid is produced enzymatically,in the absence of microbes. In some aspects, 4-(hydroxymethyl)furoicacid is produced enzymatically in one or more vessels. In some aspects,the one or more vessels are substantially free of microbes. In someaspects, the enzymatic production of 4-(hydroxymethyl)furoic acid isperformed in the same step-wise fashion as described with in the methodsutilizing recombinant microorganisms, but substantially free ofmicroorganisms or in the absence of microorganisms. In some aspects, theenzymes utilized in the enzymatic production of 4-(hydroxymethyl)furoicacid are isolated from microbes, recombinant or otherwise, and providedto their corresponding substrates for the stepwise production of theintermediates utilized to produce 4-(hydroxymethyl)furoic acid. In someaspects, one or more of the steps of the methods are performed in thesame vessel. In some aspects, once the desired product is produced as aresult of the individual method steps described herein, the product isisolated and purified and then utilized as the substrate in the nextstep of the method of producing 4-(hydroxymethyl)furoic acid.

2-formylfuran-4-carboxylate

In one embodiment, the present disclosure is drawn to a method ofproducing a recombinant microorganism capable of producing2-formylfuran-4-carboxylate from a carbon source. Some embodiments ofthe present disclosure are presented in FIG. 1 , FIG. 2 , and FIG. 3 ,which collectively detail the biosynthetic conversion of a carbonfeedstock to 2-formylfuran-4-carboxylate.

In one embodiment, step F in FIG. 2 is a single step reaction utilizingfuran-2,4-dicarbaldehyde as a substrate. In one embodiment, thebioproduction of 2-formylfuran-4-carboxylate fromfuran-2,4-dicarbaldehyde is catalyzed by one or more enzymes representedby EC numbers 1.2.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion offuran-2,4-dicarbaldehyde to 2-formylfuran-4-carboxylate. In oneembodiment, the dehydrogenase is classified as EC number 1.2.1. In oneembodiment, the dehydrogenase EC number 1.2.1 selected from aldehydedehydrogenase (NAD+) (EC number 1.2.1.3) or aldehyde dehydrogenase(NADP+) (EC number 1.2.1.4) or aldehyde dehydrogenase [NAD(P)+] (ECnumber 1.2.1.5) or 4-(γ-glutamylamino)butanal dehydrogenase (EC number1.2.1.99). In one embodiment, the oxidase is classified as EC number1.1.3. In one embodiment, the oxidase EC number 1.1.3 is5-(hydroxymethylfurfural oxidase (EC number 1.1.3.47). In someembodiments the EC 1.1.3.47 oxidase can be derived from the gene hmfH.In some embodiments, hmfH can be derived from Methylovorus sp. MP688 orCupriavidus basilensis. In one embodiment, the oxidase EC number 1.1.3is aryl-alcohol oxidase (EC number 1.1.3.7). In one embodiment, theperoxygenase is classified as EC number 1.11.2. In one embodiment, theperoxygenase EC number 1.11.2 is unspecific peroxygenase (EC number1.11.2.1).

In some aspects, 2-formylfuran-4-carboxylate is produced enzymatically,in the absence of microbes. In some aspects, 2-formylfuran-4-carboxylateis produced enzymatically in one or more vessels. In some aspects, theone or more vessels are substantially free of microbes. In some aspects,the enzymatic production of 2-formylfuran-4-carboxylate is performed inthe same step-wise fashion as described with in the methods utilizingrecombinant microorganisms, but substantially free of microorganisms orin the absence of microorganisms. In some aspects, the enzymes utilizedin the enzymatic production of 2-formylfuran-4-carboxylate are isolatedfrom microbes, recombinant or otherwise, and provided to theircorresponding substrates for the stepwise production of theintermediates utilized to produce 2-formylfuran-4-carboxylate. In someaspects, one or more of the steps of the methods are performed in thesame vessel. In some aspects, once the desired product is produced as aresult of the individual method steps described herein, the product isisolated and purified and then utilized as the substrate in the nextstep of the method of producing 2-formylfuran-4-carboxylate.

4-formylfuran-2-carboxylate

In one embodiment, the present disclosure is drawn to a method ofproducing a recombinant microorganism capable of producing4-formylfuran-2-carboxylate from a carbon source. Some embodiments ofthe present disclosure are presented in FIG. 1 , FIG. 2 , and FIG. 3 ,which collectively detail the biosynthetic conversion of a carbonfeedstock to 4-formylfuran-2-carboxylate.

In one embodiment, step G in FIG. 2 is a single step reaction utilizingfuran-2,4-dicarbaldehyde as a substrate. In one embodiment, thebioproduction of 4-formylfuran-2-carboxylate fromfuran-2,4-dicarbaldehyde is catalyzed by one or more enzymes representedby EC numbers 1.2.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion offuran-2,4-dicarbaldehyde to 4-formylfuran-2-carboxylate. In oneembodiment, the dehydrogenase is classified as EC number 1.2.1. In oneembodiment, the dehydrogenase EC number 1.2.1 selected from aldehydedehydrogenase (NAD+) (EC number 1.2.1.3) or aldehyde dehydrogenase(NADP+) (EC number 1.2.1.4) or aldehyde dehydrogenase [NAD(P)+] (ECnumber 1.2.1.5) or 4-(γ-glutamylamino)butanal dehydrogenase (EC number1.2.1.99). In one embodiment, the oxidase is classified as EC number1.1.3. In one embodiment, the oxidase EC number 1.1.3 is5-(hydroxymethylfurfural oxidase (EC number 1.1.3.47). In someembodiments the EC 1.1.3.47 oxidase can be derived from the gene hmfH.In some embodiments, hmfH can be derived from Methylovorus sp. MP688 orCupriavidus basilensis. In one embodiment, the oxidase EC number 1.1.3is aryl-alcohol oxidase (EC number 1.1.3.7). In one embodiment, theperoxygenase is classified as EC number 1.11.2. In one embodiment, theperoxygenase EC number 1.11.2 is unspecific peroxygenase (EC number1.11.2.1).

In one embodiment, step H in FIG. 2 is a single step reaction utilizing4-(hydroxymethyl)furoic acid as a substrate. In one embodiment, thebioproduction of 4-formylfuran-2-carboxylate from4-(hydroxymethyl)furoic acid is catalyzed by one or more enzymesrepresented by EC numbers 1.1.1.-, 1.1.3.-, and 1.11.2.-.

In one embodiment, the recombinant microorganism of any one of theembodiments disclosed herein comprises at least one endogenous orexogenous nucleic acid molecule encoding a dehydrogenase, or a oxidase,or a peroxygenase that catalyzes the conversion of4-(hydroxymethyl)furoic acid to 4-formylfuran-2-carboxylate. In oneembodiment, the dehydrogenase is classified as EC number 1.1.1. In oneembodiment, the dehydrogenase EC number 1.1.1 selected from alcoholdehydrogenase (EC number 1.1.1.1), or alcohol dehydrogenase (NADP+) (ECnumber 1.1.1.2), or D-xylose reductase (EC number 1.1.1.307), oraryl-alcohol dehydrogenase (EC number 1.1.1.90), or aryl-alcoholdehydrogenase (NADP+) (EC number 1.1.1.91). In one embodiment, theoxidase is classified as EC number 1.1.3. In one embodiment, the oxidaseEC number 1.1.3 is 5-(hydroxymethylfurfural oxidase (EC number1.1.3.47). In some embodiments the EC 1.1.3.47 oxidase can be derivedfrom the gene hmfH. In some embodiments, hmfH can be derived fromMethylovorus sp. MP688 or Cupriavidus basilensis. See Dijkman andFraaije (2014) and Koopman et al. (2010). In one embodiment, the oxidaseEC number 1.1.3 is aryl-alcohol oxidase (EC number 1.1.3.7). See Carroet al. (2015). In one embodiment, the peroxygenase is classified as ECnumber 1.11.2. In one embodiment, the peroxygenase EC number 1.11.2 isunspecific peroxygenase (EC number 1.11.2.1). See Carro et al. (2015).

In some aspects, 4-formylfuran-2-carboxylate is produced enzymatically,in the absence of microbes. In some aspects, 4-formylfuran-2-carboxylateis produced enzymatically in one or more vessels. In some aspects, theone or more vessels are substantially free of microbes. In some aspects,the enzymatic production of 4-formylfuran-2-carboxylate is performed inthe same step-wise fashion as described with in the methods utilizingrecombinant microorganisms, but substantially free of microorganisms orin the absence of microorganisms. In some aspects, the enzymes utilizedin the enzymatic production of 4-formylfuran-2-carboxylate are isolatedfrom microbes, recombinant or otherwise, and provided to theircorresponding substrates for the stepwise production of theintermediates utilized to produce 4-formylfuran-2-carboxylate. In someaspects, one or more of the steps of the methods are performed in thesame vessel. In some aspects, once the desired product is produced as aresult of the individual method steps described herein, the product isisolated and purified and then utilized as the substrate in the nextstep of the method of producing 4-formylfuran-2-carboxylate.

The present disclosure provides methods and recombinant microorganismscapable of producing high yields of one or more of 4-HMF,2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoicacid, 2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and2,4-FDCA. In one embodiment one molecule of glucose and two molecules ofATP are converted into one molecule of 2,4-FDCA and three molecules ofNAD(P)H according to the net equation 1:1 glucose+2 ATP→1 2,4-FDCA+3 NAD(P)H  Equation 1

The net reaction results in a mass yield of about 0.87 grams of 2,4-FDCAper gram of glucose. This yield is equivalent to 75% of the maximalthermodynamic yield of 1.16 grams of 2,4-FDCA per gram of glucose. Insome embodiments, the yield of 2,4-FDCA can be about 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.16 grams per gram of glucose.

In one embodiment one molecule of glucose, two molecules of ATP andthree molecules of oxygen are converted into one molecule of 2,4-FDCAand three molecules of hydrogen peroxide (H₂O₂) according to the netequation 2:1 glucose+2 ATP+3 O₂→1 2,4-FDCA+3 H₂O₂  Equation 2

In one embodiment two molecules of glycerol and two molecules of ATP areconverted into one molecule of 2,4-FDCA and five molecules of NAD(P)Haccording to the net equation 3:2 glycerol+2 ATP→1 2,4-FDCA+5 NAD(P)H  Equation 3

The net reaction results in a mass yield of about 0.85 grams of 2,4-FDCAper gram of glycerol. This yield is equivalent to 64% of the maximalthermodynamic yield of 1.32 grams of 2,4-FDCA per gram of glycerol. Insome embodiments, the yield of 2,4-FDCA can be about 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.32 grams per gram of glycerol.

In one embodiment two molecules of glycerol, two molecules of ATP andthree molecules of oxygen are converted into one molecule of 2,4-FDCAand three molecules of hydrogen peroxide according to the net equation4:2 glycerol+2 ATP+3 O₂→1 2,4-FDCA+3 H₂O₂  Equation 4Methods of Producing and Isolating Biosynthesis Product Monomers and/orPolymers

In some aspects, modified microbes of the present disclosure aremodified such that the microbes produce 4-HMF, 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and 2,4-FDCAmonomers. In some aspects, modified microbes of the present disclosureare modified such that the microbes produce polymers derived from 4-HMF,2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoicacid, 2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and2,4-FDCA. In some aspects, a method of producing the biosynthesisproduct monomers and/or polymers comprises growing/fermenting one ormore microbes of the present disclosure under conditions sufficient toproduce the biosynthesis product monomers and/or polymers, andisolating/collecting the resulting 4-HMF, 2,4-furandimethanol,furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and 2,4-FDCAand/or polymers thereof. In some aspects, the biosynthesis monomers willpolymerize into polymers in vivo. In some aspects, the production of thebiosynthesis monomers and polymers is proportional to the number ofbacteria utilized in the microbial fermentation process. In someaspects, the bacteria are grown in a reaction chamber. Once a desirednumber of bacteria have been achieved, the spent media is subjected to aprocess for the isolating the biosynthesis product monomers and/orpolymers. In some aspects, the microbes are lysed and the cellulardebris is pelleted out of solution in a centrifuge. In some aspects, thebiosynthesis product monomers and/or polymers are collected from thecell pellet fraction or the liquid fraction with the aid of a solventextraction process or a gradient ultra-centrifugation process. In someaspects, the biosynthesis product polymer can be isolated by filtration.

In some aspects, a biosynthesis product monomer is produced bycultivating the recombinant microorganism in a culture medium containinga feedstock providing a carbon source until the monomer is produced. Insome aspects, the feedstock comprises one or more hexose, one or morepentose, or a combination thereof. In some aspects, the monomer isextracted from the culture medium and polymerized in the presence of acatalyst. The present disclosure provides a method of producing apolymer from biosynthesis product produced by the recombinantmicroorganisms and methods of the disclosure. In one embodiment the oneor more biosynthesis products are catalytically polymerized with a diolto form a polymer. The diol can be selected from ethylene glycol,1,3-propanediol, 1,2-propanediol, 1,4-butanediol, 1,3-butanediol,2,3-butanediol, 1,5-pentanediol, or 1,6-hexanediol.

In some aspects, the biosynthesis product monomer is catalyzed in thepresence of a catalyst selected from a titanium-based catalyst,germanium-based catalyst, magnesium-based catalyst, silicon-basedcatalyst, aluminum-based catalyst, or an antimony-based catalyst. Insome aspects, the catalyst is selected from: antimony acetate, antimonytrioxide, germanium dioxide, tetra-isopropyl titanate, and tetra-n-butyltitanate.

In some aspects, the biosynthesis product-derived polymer is polymerizedin vivo by a pha synthase. In some aspects, the biosynthesisproduct-derived monomer is polymerized ex vivo by a pha synthase.

In some aspects, the biosynthesis product 4-HMF is extracted from theculture medium and transformed, in the presence of a catalyst, into oneor more of the other biosynthesis products as reported in the state ofthe art. See Van Putten et al. (2013. Hydroxymethylfurfural, a VersatilePlatform Chemical Made from Renewable Resources. Chemical Reviews,113.3:1499-1597).

In some aspects, the biosynthesis product 4-HMF is extracted from theculture medium and transformed, in the presence of a catalyst, into2,4-dimethylfuran. See Deng et al. (2013. Linked Strategy for theProduction of Fuels via Formose Reaction. Scientific Reports, 3:1244).

In some aspects, any one or more of the biosynthesized products producedby the methods and compositions described herein are extracted from theculture medium in which they are biosynthesized and are transformed inthe presence of a chemical or biological catalyst(s).

In some aspects, the transformation of the biosynthesized products inthe presence of a chemical or biological catalyst(s) is performed in theabsence of microorganisms. In some aspects, the the transformation ofthe biosynthesized products in the presence of a biological catalyst(s)is performed in the absence of microorganisms and in the presence of oneor more enzymes isolated and purified from one or more microorganisms.

In some aspects, the chemical catalyst or catalysts are any one or moreof the chemicals that are known to be utilized in non-biologicalsynthesis of 2,4-furandimethanol, furan-2,4-dicarbaldehyde,4-(hydroxymethyl)furoic acid, 2-formylfuran-4-carboxylate,4-formylfuran-2-carboxylate, and/or 2,4-FDCA.

In some aspects, the biological catalyst or catalysts are any one ormore of the enzymes described herein for reaction steps C, D, E, F, G,H, I, or J in FIG. 2 .

In some aspects, the transformation of biosynthesized 4-HMF into2,4-furandimethanol occurs in a composition substantially free ofmicroorganisms and in the presence of a chemical or biological catalystdescribed herein, such as the biological catalysts of EC 1.1.1.-.

In some aspects, the transformation of biosynthesized 4-HMF intofuran-2,4-dicarbaldehyde occurs in a composition substantially free ofmicroorganisms and in the presence of a chemical or biological catalystdescribed herein, such as the biological catalysts of EC 1.1.1.-, EC1.1.3.-, and/or EC 1.11.2.-.

In some aspects, the transformation of biosynthesized 4-HMF into4-(hydroxymethyl)furoic acid) occurs in a composition substantially freeof microorganisms and in the presence of a chemical or biologicalcatalyst described herein, such as the biological catalysts of EC1.1.1.-, EC 1.1.3.-, and/or EC 1.11.2.-.

In some aspects, the transformation of biosynthesized2,4-furandimethanol into 2-formylfuran-4-carboxylate occurs in acomposition substantially free of microorganisms and in the presence ofa chemical or biological catalyst described herein, such as thebiological catalysts of EC 1.2.1.-, EC 1.1.3.-, and/or EC 1.11.2.-.

In some aspects, the transformation of biosynthesizedfuran-2,4-dicarbaldehyde into 2-formylfuran-4-carboxylate occurs in acomposition substantially free of microorganisms and in the presence ofa chemical or biological catalyst described herein, such as thebiological catalysts of EC 1.2.1.-, EC 1.1.3.-, and/or EC 1.11.2.-.

In some aspects, the transformation of biosynthesizedfuran-2,4-dicarbaldehyde into 4-formylfuran-2-carboxylate occurs in acomposition substantially free of microorganisms and in the presence ofa chemical or biological catalyst described herein, such as thebiological catalysts of EC 1.2.1.-, EC 1.1.3.-, and/or EC 1.11.2.-.

In some aspects, the transformation of biosynthesized4-(hydroxymethyl)furoic acid into 4-formylfuran-3-carboxylate occurs ina composition substantially free of microorganisms and in the presenceof a chemical or biological catalyst described herein, such as thebiological catalysts of EC 1.2.1.-, EC 1.1.3.-, and/or EC 1.11.2.-.

In some aspects, the transformation of biosynthesized2-formylfuran-4-carboxylate into 2,4-FDCA occurs in a compositionsubstantially free of microorganisms and in the presence of a chemicalor biological catalyst described herein, such as the biologicalcatalysts of EC 1.2.1.-, EC 1.1.3.-, and/or EC 1.11.2.-.

In some aspects, the transformation of biosynthesized4-formylfuran-2-carboxylate into 2,4-FDCA occurs in a compositionsubstantially free of microorganisms and in the presence of a chemicalor biological catalyst described herein, such as the biologicalcatalysts of EC 1.2.1.-, EC 1.1.3.-, and/or EC 1.11.2.-.

In some aspects, any one or more of the transformations described abovecan be combined with another transformation such that the product of thefirst transformation is the substrate for the product of the secondtransformation.

In some aspects, any one or more of the transformations described abovecan be combined with another transformation such that the product of thefirst transformation is the substrate for the product of the secondtransformation, whose product is the substrate for the product of thethird transformation.

Biological Processes for Producing the Biosynthesis Products

The present disclosure provides a biological process for producing oneor more of the biosynthesis products described herein; 4-HMF,2,4-furandimethanol, furan-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoicacid, 2-formylfuran-4-carboxylate, 4-formylfuran-2-carboxylate, and2,4-FDCA. In some embodiments, the process comprises: providing to atleast one bioreactor one or more recombinant microorganisms engineeredto express one or more enzymes involved in the biosynthesis ofglyceraldehyde 3-phosphate (G3P) from one or more biosynthesis pathwaysand one or more of the biosynthesis products from G3P and a feedstockcomprising an exogenous carbon source; cultivating the one or morerecombinant microorganisms in one or more stages in a culture mediumcomprising the feedstock; fermenting the resulting culture in one ormore stages under aerobic, microaerobic and/or anaerobic conditions; andrecovering from the bioreactor the one or more biosynthesis productsafter the fermentation step.

In some embodiments of the biological process, the one or morebiosynthesis products are recovered continuously prior to exhaustion ofthe culture medium or the feedstock. In some embodiments, thebiosynthesis products are recovered in batches prior to exhaustion ofthe culture medium or the feedstock. In some embodiments, the one ormore recombinant microorganisms are derived from a parentalmicroorganism selected from the group consisting of Clostridium sp.,Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridiumragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum,Moorella thermoacetica, Corynebacterium glutamicum, Clostridiumaceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridiumdrakei, Clostridium carboxidivorans, Clostridium formicoaceticum,Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum,Blautia producta, Clostridium glycolicum, Clostridium magnum, Candidakrusei, Clostridium mayombei, Clostridium methoxybenzovorans,Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacterpfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogeniumphaeum, Acetobacterium carbinolicum, Issatchenkia orientalis, Sporomusatermitida, Moorella glycerini, Eubacterium aggregans, Treponemaazotonutricium, Pichia kudriavzevii, Escherichia coli, Saccharomycescerevisiae, Pseudomonas putida, Bacillus sp, Corynebacterium sp.,Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacterglycolicus.

In some embodiments of the biological process, the feedstock comprisesC6 carbohydrates and/or C5 carbohydrates. In some embodiments, thefeedstock comprises monosaccharides, disaccharides, oligosaccharides,polysaccharides, or combinations thereof.

In some embodiments of the biological process, the cultivating andfermenting steps occur in the same stage. In some embodiments, thecultivating and fermenting steps occur in separate stages. In someembodiments, the cultivating and fermenting steps occur in separatebioreactors. In some embodiments, the cultivating and fermenting stepsoccurs in the same bioreactor. In some embodiments, the bioreactoroperates under aerobic, microaerobic, or anaerobic conditions; or acombination thereof.

In some embodiments, the one or more stages receive the culture and/orculture media as a batch, a fed-batch, or a continuous mode feed. Insome embodiments, the cultivating stage receives the culture and/orculture media as a batch, a fed-batch, or a continuous mode feed, andany subsequent stages operate as a batch, a fed-batch, or a continuousmode feed.

In some embodiments, the culture medium comprises carbon (C) that isprovided from C5 carbohydrates, C6 carbohydrates, and/or disaccharides.In some embodiments, the culture medium comprises essential nutrientsincluding nitrogen (N), phosphorus (P), magnesium (Mg), and iron (Fe).

In some embodiments, wherein a ratio of C:N in the cultivating step isat least 10:1. In some embodiments, wherein a ratio of C:P in thecultivating step is at least 5:1. In some embodiments, a ratio of C:Mgin the cultivating step is at least 50:1. In some embodiments, a ratioof C:Fe in the cultivating step is at least 300:1.

In some embodiments, the cultivating step operates from 5 up to 100hours for the cultivation of the cells of the one or more recombinantmicroorganisms. In some embodiments, the culture in the fermenting stepcomprises about 1% to about 30% of the cell mass, which is transferredfrom the cultivating step in the culture medium with the one or moresubstrates. In some embodiments, a total amount of the feedstockprovided to the fermenting step ranges from about 100 kg/m³ to about 800kg/m³.

In some embodiments, a ratio of C:N in the fermenting step is at least50:1. In some embodiments, a ratio of C:P in the fermenting step is atleast 20:1. In some embodiments, a ratio of C:Mg in the fermenting stepis at least 200:1. In some embodiments, a ratio of C:Fe in thefermenting step is at least 800:1. In some embodiments, the fermentingstep operates from 10 up to 300 hours for fed-batch operation and up to300 hours for continuous operation.

EXAMPLES Example 1: Expression and Purification of Methyl PhosphateSynthase

The expression and purification of enzymes used in enzymatic assays wascarried out under the following conditions: Genes coding 27(5-formylfuran-3-yl)methyl phosphate synthases candidates (Table 1) weresynthetized by GenScript and cloned in expression vector pET28a in NdeIand BamHI restriction sites. The expression vector was transformed intoE. coli BL21 (DE3) and the transformant was stored in 15% glycerol untiluse for enzyme expression.

The stored transformant was inoculated into 50 mL of TB broth containingkanamycin at 37° C. with agitation for 16 h to prepare a seed culture.The seed culture was added to 300 mL of TB broth containing kanamycinwith initial OD (600 nm) of 0.2, the culture was then incubated at 37°C. with agitation until OD (600 nm) reached 0.6-0.8 at which point 1 mMIPTG was added to induce expression overnight at 18° C. with agitation.

Following overnight expression, the cells were centrifuged at 6000× rpmfor 30 min and the pellet cell was suspended in cold lysis buffer (20 mMphosphate buffer and 500 mM NaCl pH 7.4) before ultrasonic disruption.The cell lysate was again centrifuged at 8000 rpm for 30 min at 4° C.and filtered before purification with affinity chromatography. Thecolumn utilized was a HisTrap FF Crude (GE Healthcare) for his-taggedprotein purification. The purified protein was bound and washed in thecolumn with binding buffer A (20 mM phosphate buffer, 20 mM imidazole,500 mM NaCl, 1 mM PMSF and beta-mercaptoethanol, pH 7.4) and eluted in agradient of elution buffer B (20 mM phosphate buffer, 500 mM imidazole,500 mM NaCl, 1 mM PMSF and beta-mercaptoethanol, pH 7.4). Then using aPD-10 column the buffer was changed to a 50 mM Tris-HCl pH 7.4.Candidates expression and purification were analyzed on 12%polyacrylamide gel by electrophoresis, as illustrated at FIG. 4 .

Example 2: (5-formylfuran-3-yl)methyl phosphate Production from G3P

The (5-formylfuran-3-yl)methyl phosphate production fromglyceraldehyde-3-phosphate (G3P) by enzyme candidates described in Table1 was demonstrated in vitro by incubating approximately 450 μg ofpurified candidates with a 1 mL solution containing 5 mM ofglyceraldehyde-3-phosphate (Sigma) in 20 mM Tris-HCl, 200 mM NaCl (pH7.4) buffer. The reaction was incubated at 37° C. for 2 hours. Reactionvessels without synthases or substrate (G3P) were used as negativecontrols. The reaction was monitored by UV-Vis using a spectrophotometer(SpectraMax M5, Molecular Devices), accordingly to the5-formylfuran-3-yl)methyl phosphate Molar absorption coefficient (c) 280nm. Product formation was also confirmed by HPLC analysis.

The chromatographic quantitative analysis of (5-formylfuran-3-yl)methylphosphate production was performed in a HPLC-DAD (Thermo Ultimate 3000)equipped with an Aminex HPX-87H Biorad column (300×7.8 mm). The columnwas maintained at 50° C. and the mobile phase used was a 5 mM H₂SO₄solution with flow rate of 0.75 mL/min (isocratic gradient mode).

As shown in Table 6, FIG. 5 , and FIG. 6 , G3P was successfullyconverted into (5-formylfuran-3-yl)methyl phosphate by methyl phosphatesynthases. In FIG. 5 , the negative control sample (grey line) shows alow absorbance at 280, indicating little to no presence of the(5-formylfuran-3-yl)methyl phosphate in comparison to the reactioncontaining the methyl phosphoate synthase (black line), indicating thatthe synthase catalyzed the formation of the (5-formylfuran-3-yl)methylphosphate from G3P; these results are summarized in Table 6. FIG. 6shows detectable (5-formylfuran-3-yl)methyl phosphate in the reactioncontaining synthase (Lower Panel) but not in the negative controlreaction (Upper Panel). Table 7 contains a list of methyl phosphatesynthase candidates that positively tested for the production of(5-formylfuran-3-yl)methyl phosphate from G3P.

TABLE 6 Absorbance obtained at 280 nm for methyl phosphate synthaseproduction of (5-formylfuran-3-yl)methyl phosphate from G3P. Absorbanceat 280 nm Methyl phosphate synthase positive reaction 2.97 (MfnB1candidate) Negative control 0.26

TABLE 7 (5-formylfuran-3-yl)methyl phosphate synthases candidates thatpositively tested for catalyzing the production of(5-formylfuran-3-yl)methyl phosphate production from G3P. Name OrganismMfnB 1 Methanocaldococcus jannaschii MfnB 2 Methanocaldococcus fervensMfnB 3 Methanocaldococcus vulcanius MfnB 4 Methanocaldococcus infernosMfnB 5 Methanothermococcus okinawensis MfnB 6 Methanococcales archaeonHHB MfnB 7 Methanobrevibacter smithii MfnB 8 Methanobacterium sp.PtaB.Bin024 MfnB 9 Methanopyrus sp. KOL6 MfnB 10 Candidatus Argoarchaeumethanivorans MfnB 12 Methanobrevibacter arboriphilus MfnB 13Methanococcus maripaludis MfnB 14 Methanococcus vannielii MfnB 15Methanosarcina acetivorans MfnB 16 Methanosarcina barkeri MfnB 17Methylorubrum extorquens MfnB 18 Methylobacterium sp. MfnB 19Methanosarcina mazei MfnB 20 Methyloversatilis universalis MfnB 22Streptomyces cattleya NRRL 8057 MfnB 23 Streptomyces coelicolor MfnB 24Streptomyces EFF88969 MfnB 25 Streptomyces gris{acute over (e)}us MfnB26 Streptomyces sp. DH-12 MfnB 27 Streptomyces venezuelae

Example 3: Production of 4-hydroxymethylfurfural (4-HMF) from(5-formylfuran-3-yl)methyl phosphate

The production of 4-HMF from (5-formylfuran-3-yl)methyl phosphate usingphosphatases was demonstrated using commercially available phosphatase,E. coli lysates, and yeast lysates to demonstrate their capability toproduce 2,4-HMF from (5-formylfuran-3-yl)methyl phosphate. The substrate(5-formylfuran-3-yl)methyl phosphate was produced by(5-formylfuran-3-yl)methyl phosphate synthases as described at Example2.

The chromatographic quantitative analysis of (5-formylfuran-3-yl)methylphosphate and 4-HMF production was performed in a HPLC-DAD (ThermoUltimate 3000) equipped with an Aminex HPX-87H Biorad column (300×7.8mm). The column was maintained at 50° C. and the mobile phase used was a5 mM H₂SO₄ solution with flow rate of 0.75 mL/min (isocratic gradientmode). Both compounds were detected at 280 nm.

To carry out the reaction demonstrating the production of 4-HMF from(5-formylfuran-3-yl)methyl phosphate using a commercially availablephosphatase, 2 μL of alkaline phosphatase from bovine intestinal mucosa(Sigma) was added to 1 mL of reaction vessel from Example 2, containingapproximately 1-2 mM of (5-formylfuran-3-yl)methyl phosphate. Thereaction was incubated at 37° C. for 1 h and initial and final sampleswere analyzed by HPLC-DAD. As shown in FIG. 7 (Upper Panel) and Table 8,the commercially available phosphatase was able to perform the fullconversion of (5-formylfuran-3-yl)methyl phosphate to 4-HMF.

TABLE 8 Peak area of (5-formylfuran-3-yl)methyl phosphate produced usingmethyl phosphate synthase. Area (mAU*min) Methyl phosphate synthasepositive reaction 62,1544 Negative control 0

To carry out the reaction demonstrating the production of 4-HMF from(5-formylfuran-3-yl)methyl phosphate using phosphatases in an E. colilysate, a strain of E. coli MG1655 was inoculated into 200 mL of LBbroth at 37° C. with agitation overnight. The culture was centrifuged at4000 rpm for 15 min and the pellet suspended in 20 mL of 20 mM HEPESbuffer pH 7.4 resulting in an OD of 70. The lysis was performed byultrasonic disruption. 1 mL of the E. coli lysate was mixed with 1 mL ofreaction from Example 2 and incubated overnight at 37° C. withagitation. Samples were analyzed by HPLC-DAD at 280 nm for production of4-HMF.

To carry out the reaction demonstrating the production of 4-HMF from(5-formylfuran-3-yl)methyl phosphate using phosphatases in a yeastlysate, a strain of Saccharomyces cerevisiae was inoculated into 200 mLof YPD broth at 30° C. with agitation overnight. The culture wascentrifuged at 4000 rpm for 15 min and the pellet suspended in 20 mL of20 mM HEPES buffer pH 7.4 resulting in an OD of 120. Cell lysis wasperformed by ultrasonic disruption. 1 mL of the yeast lysate was mixedwith 1 mL of reaction from example 2 and incubated overnight at 30° C.with agitation. Samples were analyzed by HPLC-DAD at 280 nm forproduction of 4-HMF.

As shown in FIG. 7 (Middle Panel) and FIG. 7 (Lower Panel) and Table 9,both E. coli and yeast lysates showed endogenous phosphatase activityable to perform the conversion of (5-formylfuran-3-yl)methyl phosphateto 4-HMF.

TABLE 9 4-HMF production from (5-formylfuran-3-yl)methyl phosphate withcommercially available phosphatase after 1 hour incubation and E. coli land yeast lysates after overnight incubation at 37° C. and 30° C.,respectively. (5-formylfuran-3-yl) 4-HMF methyl phosphate area Samplearea (mAU*min) (mAU*min) Sigma phosphatase n.a. 385,3242 E. coli lysatereaction 57,2574 5,6535 Yeast lysate reaction 187,9746 67,0542 E. colinegative control reaction n.a. n.a. (absence of 5-formylfuran-3-yl)methyl phosphate substrate) Yeast negative control reaction 22,10854,0767 (absence of 5-formylfuran-3-yl) methyl phosphate substrate)

Example 4: Expression of 4-HMF oxidases Enzymes

Genes coding 7 4-HMF oxidases enzymes candidates (Table 4) weresynthesized by GenScript and cloned in expression vector pET28a in NdeIand BamHI restriction sites. The expression vector was transformed intoE. coli BL21 (DE3) and the transformant was stored in 15% glycerol untiluse for enzyme expression.

The stored transformant was inoculated into 50 mL of TB broth containingkanamycin at 37° C. with agitation for 16 h to prepare a seed culture.The seed culture was added to 300 mL of TB broth containing kanamycinwith initial OD (600 nm) of 0.2, the culture was then incubated at 37°C. with agitation until OD (600 nm) reached 0.6-0.8 at which point 1 mMIPTG was added to induce expression overnight at 18° C. with agitation.

Following overnight expression, the cells were centrifuged at 6000× rpmfor 30 min, the cell pellet was suspended in cold lysis buffer (20 mMphosphate buffer and 500 mM NaCl pH 7.4) before ultrasonic disruption.The cell lysate was again centrifuged at 8000× rpm for 30 min at 4° C.and filtered before purification with affinity chromatography. Thecolumn utilized was a HisTrap FF Crude (GE Healthcare) for thehis-tagged protein purification. The purified protein was bound andwashed in the column with binding buffer A (20 mM phosphate buffer, 20mM imidazole, 500 mM NaCl, 1 mM PMSF and beta-mercaptoethanol, pH 7.4)and eluted in a gradient of elution buffer B (20 mM phosphate buffer,500 mM imidazole, 500 mM NaCl, 1 mM PMSF and beta-mercaptoethanol, pH7.4). Then, using a PD-10 column, the buffer was changed to a 50 mMTris-HCl pH 7.4. Candidates expression and purification were analyzed on12% polyacrylamide gel by electrophoresis, as illustrated at FIG. 8 .

Example 5: Production of 2,4-FDCA from 2,4-HMF by HmfH oxidases

The 2,4-FDCA production from 2,4-HMF by enzyme candidates described inTable 4 was demonstrated in vitro by incubating approximately 100 μg ofpurified HmfH oxidase candidates with a 1 mL of reaction vessel fromExample 3 (using the commercially available phosphatase), containingapproximately 1 mM 4-HMF. The reaction was incubated at 30° C. for 16hours and both initial and final samples analyzed by HPLC-DAD. Sampleswere injected in HPLC-DAD and the production of 2,4-FDCA and itsintermediates confirmed by GC-MS.

The quantitative analysis of 2,4-FDCA was performed using HPLC-DAD(Thermo Ultimate 3000) equipped with an Aminex HPX-87H Biorad column(300×7.8 mm). The column was maintained at 50° C. The mobile phase usedwas a 5 mM H₂SO₄ solution with flow rate of 0.6 mL/min with isocraticgradient mode. The molecule was detected at 245 nm.

For GC-MS identification, initial and final samples were stopped byadding 6M HCl to reduce pH to 2-3. The products were liquid/liquidextracted using ethyl acetate and dried with Na₂SO₄ to remove watertraces. The extracted material was then evaporated in a speedvac andderivatized using bis-(trimethylsilyl)trifluoroacetamide at 60° C. for 2h. The samples were injected in a gas chromatograph with HP-5MS column(Agilent, 30 m×0.25 mm ID, 0.25 um film thickness) coupled with aquadrupole mass detector (ISQ, Thermo). The oven program started at 110°C. for 2 min with increasing ramp of 20° C./min until 300° C. that washeld for 3 min. Helium was used as carrier gas at a flow rate of 1.2mL/min. 2,4-FDCA was identified by comparing their mass spectra withthose in literature. (Ref: Carro, Juan, et al. “5-hydroxymethylfurfuralconversion by fungal aryl-alcohol oxidase and unspecific peroxygenase.”The FEBS journal 282.16 (2015): 3218-3229.) 4-formylfuran-2-carboxylate(2,4-FFCA) and furan-2,4-dicarbaldehyde (2,4-DFF) were also identifiedby their mass spectra.

As shown in Table 10 and FIG. 9 , FIG. 10 , and FIG. 11 , the conversionof 4-HMF into 2,4-FDCA was successfully demonstrated with 4-HMFoxidases, especially with enzyme HmfH7 that was able to fully convert2,4-HMF into 2,4-FDCA.

FIG. 9 (Upper Panel) shows the chromatogram of HmfH1, FIG. 9 (MiddlePanel) the chromatogram of HmfH6 and FIG. 9 (Lower Panel) thechromatogram of HmfH7. FIG. 10 (Upper Pannel) shows the relativeabundance of the products obtained in GC-MS and the mass spectra (LowerPanel) of silylated 2,4-FDCA (FIG. 11 ).

TABLE 10 2,4 FDCA production from 4-HMF with 4-HMF oxidases candidatesafter 16 hours incubation. The reaction intermediates 4-formylfuran-2-carboxylate (2,4-FFCA) and furan-2,4-dicarbaldehyde (2,4-DFF) werealso identified and quantified^(a). 2,4-HMF 2,4-FDCA 2,4-FFCA 2,4-DFFReaction area area area area Condition (mAU*min) (mAU*min) (mAU*min)(mAu*min) negative 100 n.a. n.a. n.a. control reaction HmfH1 n.a.34,2154 42,5550 n.a. HmfH6 1,4416 63,9778 8,1581 1,5700 HmfH7 n.a.93,0784 n.a. n.a. ^(a)Negative control reaction was performed in similarassay condition but in absence of HMF-oxidase enzymes.

Example 6: Production of 2,4-furandimethanol from 4-HMF—Reaction C

Purified enzymes were produced as described at Example 1.

Production of 2,4-furandimethanol from 4-HMF by enzyme candidatesdescribed in Table 5 was demonstrated in vitro by incubatingapproximately 20 μg of purified enzyme candidates in 100 mM potassiumphosphate buffer (pH 7) with 0.5 mM NAD(P)H or NADH. The reactions werestarted by the addition of 0.5 mM 4-HMF obtained as shown in Example 3.The decrease of NAD(P)H was monitored at 340 nm during 40 min at 37° C.on a UV-Vis spectrophotometer (SpectraMax M5, Molecular Devices).Product formation was also confirmed by HPLC and GC-MS analysis (Datanot shown). Reaction vessels without enzymes or substrate (4-HMF) wereused as negative controls.

As demonstrated in FIG. 12 , enzymes candidates DH1, DH2 and DH6promoted reduction of 2,4-HMF to 2,4-furandimethanol, measured by itsoxidation of NAD(P)H to NAD(P)+.

Example 7: Production of furan-2,4-dicarbaldehyde from 4-HMF—Reaction D

Purified enzymes were produced as described in Example 1. Thefuran-2,4-dicarbaldehyde production from 4-HMF by enzyme candidatesdescribed at Table 3 was demonstrated in vitro by incubatingapproximately 20 μg of purified enzyme candidates in 100 mM potassiumphosphate buffer (pH 7) with 0.5 mM NAD(P)+ or NAD+. The reactionsstarted by the addition of 0.5 mM 4-HMF obtained as shown in Example 3.The increase of NAD(P)H was monitored at 340 nm during 40 min at 37° C.on a UV-Vis spectrophotometer (SpectraMax M5, Molecular Devices).Product formation was also confirmed by HPLC and GC-MS analysis (Datanot shown). Reaction vessels without enzymes or substrate (4-HMF) wereused as negative controls.

As demonstrated for enzyme DH2, selected dehydrogenases are able tooxidate 4-HMF to furan-2,4-dicarbaldehyde in vitro. The data shown inFIG. 13 was plotted after subtraction of the baseline signal andhighlights the absorbance increase and consequently the reduction ofNAD(P)H and oxidation of 2,4-HMF to furan-2,4-dicarbaldehyde when usingthe enzyme DH2.

Example 8: Production of 4-(hydroxymethyl)furoic acid from2,4-HMF—Reaction E

Purified aldehyde dehydrogenase enzymes were produced as described atExample 1. The 4-(hydroxymethyl)furoic acid production from 4-HMF byaldehyde dehydrogenase candidates described at Table 3 was demonstratedin vitro by incubating approximately 20 μg of purified enzyme candidatesin 100 mM potassium phosphate buffer (pH 7) with 0.5 mM NAD(P)+ or NAD+.The reactions started by the addition of 0.5 mM 4-HMF obtained as shownin example 3. The increase of NAD(P)H was monitored at 340 nm during 40min at 37° C. on a UV-Vis spectrophotometer (SpectraMax M5, MolecularDevices). Product formation was also confirmed by HPLC and GC-MSanalysis (Data not shown). Reaction vessels without enzymes or substrate(4-HMF) were used as negative controls.

As representatively demonstrated for enzymes DH8, DH9, DH10 and DH11,selected aldehyde dehydrogenases are able to oxidate 4-HMF to4-(hydroxymethyl)furoic acid in vitro (FIG. 14 ). The data shown in FIG.14 was plotted after subtraction of the baseline signal and highlightsthe absorbance increase and consequently the reduction of NAD(P)H andoxidation of 2,4-HMF to 4-(hydroxymethyl)furoic acid when using therespected aldehyde dehydrogenases.

Example 9: One Pot Reaction for the Production of 2,4-FDCA from 4-HMF

Purified aldehyde dehydrogenase enzymes and alcohol dehydrogenaseenzymes were produced as described at Example 1. The one pot oxidativereaction for 2,4-FDCA production from 4-HMF was performed using DH8 asthe representative aldehyde dehydrogenase and DH6 as the representativealcohol dehydrogenase.

To carry out the reaction, 2 mL of a reaction mixture from Example 3containing 0.5 mM of 2,4-HMF and 1 mM of NAD(P)H were added 20 uM ofpurified enzyme candidates DH8 and DH6. Positive control reactions wereprepared as shown in Table 11. Two negative controls were prepared onewithout the enzymes and another one without the substrate. The reactionwas incubated at 30° C. for 16 hours and both initial and final samplesanalyzed by HPLC-DAD. Samples were injected in HPLC-DAD and theproduction of 2,4-FDCA was confirmed in GC-MS using the followingmethod.

The quantitative analysis of 2,4-FDCA was performed using HPLC-DAD(Thermo Ultimate 3000) equipped with an Aminex HPX-87H Biorad column(300×7.8 mm). The column was maintained at 50° C. The mobile phase usedwas a 5 mM H₂SO₄ solution with flow rate of 0.6 mL/min with isocraticgradient mode. The molecule was detected at 245 nm.

For GC-MS identification, initial and final samples were stopped byadding 6M HCl to reduce pH to 2-3. The products were liquid/liquidextracted using ethyl acetate and dried with Na₂SO₄ to remove watertraces. The extracted material was then evaporated in a speedvac andderivatized using bis-(trimethylsilyl)trifluoroacetamide at 60° C. for 2h. The samples were injected in a gas chromatograph with HP-5MS column(Agilent, 30 m×0.25 mm ID, 0.25 um film thickness) coupled with aquadrupole mass detector (ISQ, Thermo). The oven program started at 110°C. for 2 min with increasing ramp of 20° C./min until 300° C. that washold for 3 min. Helium was used as carrier gas at a flow rate of 1.2mL/min. 2,4-FDCA was identified by comparing their mass spectra withthose in literature. (Ref: Carro, Juan, et al. “5-hydroxymethylfurfuralconversion by fungal aryl-alcohol oxidase and unspecific peroxygenase.”The FEBS journal 282.16 (2015): 3218-3229.) 4-formylfuran-2-carboxylate(2,4-FFCA) and furan-2,4-dicarbaldehyde (2,4-DFF) were also identifiedby their mass spectra.

As shown in Table 11 and FIG. 15 (Middle Panel), the conversion of 4-HMFinto 2,4-FDCA was successfully demonstrated with the synergicaction/combination of an aldehyde dehydrogenase (DH8) and an alcoholdehydrogenase (DH6).

TABLE 11 2,4 FDCA production (2,4 FDCA peak area) from 4-HMF by thesynergic action/combination of an aldehyde dehydrogenase (DH8) and analcohol dehydrogenase (DH6) after 16 hours incubation. 2,4-FDCA areaReaction Condition (mAU*min) Positive reaction with enzymes DH8 + DH626,1823 Negative control - No enzyme 0,3357 Negative control - Nosubstrate n.a.

Example 10. In Vivo Production of 2,4-FDCA from Glucose

A plasmid containing the MfnB 1 gene (Table 1) under the control of theOXB20 promoter was constructed in a pET28a backbone. A second plasmidcontaining two 4-HMF oxidase genes (HmfH6 and HmfH7 (Table 4)) under thecontrol of OXB20 promoter was constructed in a pZS*13 backbone. Theplasmids were constructed using In-fusion commercial kit and wereconfirmed by sequencing. An E. coli K12 strain MG1655 (F-, λ-, rph-1,ilvG-, rfb-50, ΔgapA::gapN (UniProtKB-Q59931), ΔglcDEFGB, ΔaraFGH,ΔxylFGH, ΔfucO was used as production host.

The in vivo production of 2,4-FDCA from glucose was evaluated in shakeflask fermentations in triplicate, using a defined media composed by 2.2g·L⁻¹ KH₂PO₄, 9.4 g·L⁻¹ K₂HPO₄, 1.3 g·L⁻¹ (NH₄)₂SO₄, 10 mg·L⁻¹ thiamine,320 mg·L⁻¹ EDTA-NaOH, 2 mg·L⁻¹ CoCl₂.6H₂O, 10 mg·L⁻¹ MnSO₄.H₂O, 5 mg·L⁻¹CuSO₄.5H₂O, 2 mg·L⁻¹ H₃BO₃, 2 mg·L⁻¹ Na₂MoO₄.2H₂O, 54 mg·L⁻¹ ZnSO₄.7H₂O,1 mg·L⁻¹ NiSO₄.6H₂O, 100 mg·L⁻¹ citrate Fe (III), 100 mg·L⁻¹ CaCl₂.2H₂O,0.3 g·L⁻¹ MgSO₄.H₂O. Carbon source was provided by 10 g/L glucose andnitrogen sulphate was used as nitrogen source. Erlenmeyer flasks wereinoculated with the recombinant strain to an initial OD of 0.1, andincubated at 37° C., 225 rpm for 48 hours. Analysis of supernatant in 48h by HPLC indicated the production of 14±2 mg/L 2,4-FDCA (FIG. 16 ).

The invention claimed is:
 1. A method of producing 2,4-furandicarboxylicacid (2,4-FDCA) comprising fermenting a recombinant microorganism in aculture medium containing a carbon source, wherein the recombinantmicroorganism expresses the following: (a) endogenous or exogenousnucleic acid molecules capable of converting a carbon source toglyceraldehyde 3-phosphate (G3P); (b) at least one exogenous orexogenous nucleic acid molecule encoding a (5-formylfuran-3-yl)methylphosphate synthase that catalyzes the conversion of G3P from (a) to(5-formylfuran-3-yl)methyl phosphate; (c) at least one endogenous orexogenous nucleic acid molecule encoding a phosphatase that catalyzesthe conversion of (5-formylfuran-3-yl)methyl phosphate from (b) to4-hydroxymethylfurfural (4-HMF); and (d) at least one endogenous orexogenous nucleic acid molecule encoding a peroxygenase, dehydrogenase,or a oxidase that catalyzes independently or in combination theoxidation of 4-HMF from (c) to 2,4-FDCA, directly or through theproduction of intermediates furan-2,4-dicarbaldehyde,4-(hydroxymethyl)furoic acid, 4-formylfuran-2-carboxylate,4-formylfuran-2-carboxylate, and/or 2-formylfuran-4-carboxylate.
 2. Themethod of claim 1, wherein the carbon source comprises a hexose, apentose, glycerol, CO₂, sucroses or combinations thereof.
 3. The methodof claim 1, wherein the recombinant microorganism is derived from aparental microorganism selected from the group consisting of Clostridiumsp., Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridiumragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum,Moorella thermoacetica, Corynebacterium glutamicum, Clostridiumaceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridiumdrakei, Clostridium carboxidivorans, Clostridium formicoaceticum,Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum,Blautia producta, Clostridium glycolicum, Clostridium magnum, Candidakrusei, Clostridium mayombei, Clostridium methoxybenzovorans,Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacterpfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogeniumphaeum, Acetobacterium carbinolicum, Issatchenkia orientalis, Sporomusatermitida, Moorella glycerini, Eubacterium aggregans, Treponemaazotonutricium, Pichia kudriavzevii, Escherichia coli, Saccharomycescerevisiae, Pseudomonas putida, Bacillus sp, Corynebacterium sp.,Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacterglycolicus.
 4. The method of claim 1, wherein the 2,4-FDCA is recoveredor isolated by a process selected from distillation, membrane-basedseparation, gas stripping, precipitation, solvent extraction, expandedbed adsorption, or a combination thereof.
 5. A method of producing apolymer comprising: (1) fermenting a recombinant microorganism in aculture medium containing a carbon source until 2,4-furandicarboxylicacid (2,4-FDCA) monomer is produced; and (2) polymerizing the 2,4-FDCAmonomer to produce the polymer; wherein the recombinant microorganismexpresses the following: (a) endogenous or exogenous nucleic acidmolecules capable of converting a carbon source to glyceraldehyde3-phosphate (G3P); (b) at least one exogenous nucleic acid moleculeencoding a (5-formylfuran-3-yl)methyl phosphate synthase that catalyzesthe conversion of G3P from (a) to (5-formylfuran-3-yl)methyl phosphate;(c) at least one endogenous or exogenous nucleic acid molecule encodinga phosphatase that catalyzes the conversion of(5-formylfuran-3-yl)methyl phosphate from (b) to 4-hydroxymethylfurfural(4-HMF); and (d) at least one endogenous or exogenous nucleic acidmolecule encoding a peroxygenase, dehydrogenase, or a oxidase thatcatalyzes independently or in combination the oxidation of 4-HMF from(c) to 2,4-FDCA, directly or through the production of intermediatesfuran-2,4-dicarbaldehyde, 4-(hydroxymethyl)furoic acid,4-formylfuran-2-carboxylate, 4-formylfuran-2-carboxylate, and/or2-formylfuran-4-carboxylate.
 6. The method of claim 5, wherein thepolymer is a polyester.
 7. The method of claim 6, wherein the polyesteris produced from the polymerization of the 2,4-FDCA monomer with a diol.8. The method of claim 7, wherein the diol is selected from: ethyleneglycol, 1,3-propanediol, 1,2-propanediol, 1,4-butanediol,1,3-butanediol, 2,3-butanediol, 1,5-pentanediol, or 1,6-hexanediol. 9.The method of claim 6, wherein the 2,4-FDCA and a diol are catalyticallypolymerized using a catalyst in a non-biological process.
 10. The methodof claim 9, wherein the catalyst is selected from: a titanium-basedcatalyst, a germanium-based catalyst, a magnesium-based catalyst, asilicon-based catalyst, an aluminum-based catalyst, or an antimony-basedcatalyst.
 11. The method of claim 9, wherein the catalyst is selectedfrom: antimony acetate, antimony trioxide, germanium dioxide,tetra-isopropyl titanate, or tetra-n-butyl titanate.
 12. The method ofclaim 5, wherein the carbon source comprises a hexose, a pentose,glycerol, CO₂, sucroses or combinations thereof.
 13. The method of claim5, wherein the recombinant microorganism is derived from a parentalmicroorganism selected from the group consisting of Clostridium sp.,Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridiumragsdalei, Eubacterium limosum, Butyribacterium methylotrophicum,Moorella thermoacetica, Corynebacterium glutamicum, Clostridiumaceticum, Acetobacterium woodii, Alkalibaculum bacchii, Clostridiumdrakei, Clostridium carboxidivorans, Clostridium formicoaceticum,Clostridium scatologenes, Moorella thermoautotrophica, Acetonema longum,Blautia producta, Clostridium glycolicum, Clostridium magnum, Candidakrusei, Clostridium mayombei, Clostridium methoxybenzovorans,Clostridium acetobutylicum, Clostridium beijerinckii, Oxobacterpfennigii, Thermoanaerobacter kivui, Sporomusa ovata, Thermoacetogeniumphaeum, Acetobacterium carbinolicum, Issatchenkia orientalis, Sporomusatermitida, Moorella glycerini, Eubacterium aggregans, Treponemaazotonutricium, Pichia kudriavzevii, Escherichia coli, Saccharomycescerevisiae, Pseudomonas putida, Bacillus sp, Corynebacterium sp.,Yarrowia lipolytica, Scheffersomyces stipitis, and Terrisporobacterglycolicus.
 14. The method of claim 5, further comprising recovering orisolating the produced 2,4-FDCA monomer by a process selected fromdistillation, membrane-based separation, gas stripping, precipitation,solvent extraction, expanded bed adsorption, or a combination thereofprior to polymerizing the 2,4-FDCA monomer.